Erbb4 antagonists

ABSTRACT

The present invention concerns methods and means for controlling excessive proliferation and/or migration of smooth muscle cells, and in particular for treating stenosis, by using antagonists of a native ErbB4 receptor. The invention further concerns a method for the identification of ErbB4 agonists and antagonists capable of inhibiting or enhancing the proliferation or migration of smooth muscle cells.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention concerns methods and means for controllingexcessive proliferation and/or migration of smooth muscle cells, and inparticular for treating stenosis, by using antagonists of a native ErbB4receptor. The invention further concerns a method for the identificationof ErbB4 agonists and antagonists capable of inhibiting or enhancing theproliferation or migration of smooth muscle cells.

[0003] 2. Description of the Related Art

[0004] 1. ErbB Receptor Tyrosine Kinases

[0005] Transduction of signals that regulate cell growth anddifferentiation is regulated in part by phosphorylation of variouscellular proteins. Protein tyrosine kinases are enzymes that catalyzethis process. Receptor protein tyrosine kinases are believed to directcellular growth via ligand-stimulated tyrosine phosphorylation ofintracellular substrates.

[0006] HER4/Erb4 is a receptor protein tyrosine kinase belonging to theErbB family. Increased ErbB4 expression closely correlates with certaincarcinomas of epithelial origin, including breast adenocarcinomas(Plowman et al., Proc. Natl. Acad. Sci. USA 90:1746-1750 [1993]; Plowmanet al., Nature 366:473-475 [19931). Diagnostic methods for detection ofhuman neoplastic conditions (especially breast cancers) which evaluateErbB4 expression are described in EP Pat Appln No. 599,274.

[0007] Other members of the ErbB family of receptor tyrosine kinasesinclude: epidermal growth factor receptor (EGFR), ErbB2 (HER2/neu), andErbB3 (HER3). The erbB1 gene encodes the 170 kDa epidermal growth factorreceptor (EGFR) that has been causally implicated in human malignancy.In particular, increased expression of this gene has been observed inmore aggressive carcinomas of the breast, bladder, lung and stomach(Modjitahedi, H. and Dean, C. (1994) Int. J. Oncol. 4:277-296). HER4acts, in the absence of HER2, as a mediator of antiproliferative anddifferentiative response in human breast cancer cell lines. (Sartor etal., Mol. Cell Biol. 21:4265-75 (2001)).

[0008] The neu gene (also called erbB2 and HER2) encodes a 185 kDareceptor protein tyrosine kinase that was originally identified as theproduct of the transforming gene from neuroblastomas of chemicallytreated rats. Amplification and/or overexpression of the human HER2 genecorrelates with a poor prognosis in breast and ovarian cancers (Slamon,D. J. et al., Science 235:177-182 (1987); Slamon et al., Science244:707-712 (1989); and U.S. Pat. No. 4,968,603). Overexpression of HER2(frequently but not uniformly due to gene amplification) has also beenobserved in other carcinomas including carcinomas of the stomach,endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas andbladder.

[0009] A further related gene, called erbB3 or HER3, has been described.See U.S. Pat. No. 5,183,884; Kraus et al., Proc. Nail. Acad. Sci. USA86:9193-9197 (1989); EP Pat Appln No 444,961 A1; and Kraus et al., Proc.Natl. Acad. Sci. USA 90:2900-2904 (1993). Kraus et al. (1989) discoveredthat markedly elevated levels of erbB3 mRNA were present in certainhuman mammary tumor cell lines indicating that erbB3, like erbB1 anderbB2, may play a role in human malignancies. They also showed thatEGF-dependent activation of the ErbB3 catalytic domain of a chimericEGFR/ErbB3 receptor resulted in a proliferative response in transfectedNIH-3T3 cells. Furthermore, these researchers demonstrated that somehuman mammary tumor cell lines display a significant elevation ofsteady-state ErbB3 tyrosine phosphorylation further indicating that thisreceptor may play a role in human malignancies. The role of erbB3 incancer has been explored by others. It has been found to beoverexpressed in breast (Lemoine et al., Br. J. Cancer 66:1116-1121(1992)), gastrointestinal (Poller et al, J. Pathol. 168:275-280 (1992),Rajkumer et al., J. Pathol. 170:271-278 (1993), and Sanidas et al., Int.J. Cancer 54:935-940 (1993)), and pancreatic cancers (Lemoine et al., J.Pathol. 168:269-273 (1992), and Friess et al., Clinical Cancer Research1:1413-1420 (1995)). ErbB3 is unique among the ErbB receptor family inthat it possesses little or no intrinsic tyrosine kinase activity (Guyat al., Proc. Natl. Acad. Sci. USA 91:8132-8136 (1994) and Kim at al. J.Biol. Chem. 269:24747-55 (1994)).

[0010] The ErbB receptors are generally found in various combinations incells and heterodimerization is thought to increase the diversity ofcellular responses to a variety of ErbB ligands (Earp et al BreastCancer Research and Treatment 35: 115-132 (1995)). EGFR is bound by sixdifferent ligands; epidermal growth factor (EGF), transforming growthfactor alpha (TGF−), amphiregulin, heparin binding epidermal growthfactor (HB-EGF), β-cellulin and epiregulin (Groenen at al. GrowthFactors 11:235-257 (1994)). A family of heregulin proteins resultingfrom alternative splicing of a single gene are ligands for ErbB3 andErbB4. The heregulin family includes α, β and γ heregulins (Holmes atal, Science, 256:1205-1210 (1992); U.S. Pat. No. 5,641,869; and Schaeferet al. Oncogene 15:1385-1394 (1997)); neu differentiation factors(NDFs), glial growth factors (GGFs); acetylcholine receptor inducingactivity (ARIA); and sensory and motor neuron derived factor (SMOF). Fora review, see Groenen et al. Growth Factors 11:235-257 (1994); Lemke, G.Molec. & Cell. Neurosci. 7:247-262 (1996) and Lee at al. Pharm. Rev.47:51-85 (1995). Recently three additional ErbB ligands were identified;neuregulin-2 (NRG-2) which is reported to bind either ErbB3 or ErbB4(Chang et al Nature 387 509-512 (1997); and Carraway et al Nature387:512-516 (1997)); neuregulin-3 which binds ErbB4 (Zhang at al. PNAS(USA) 94(18):9562-7 (1997)); and neuregulin-4 which binds ErbB4 (Harariat al Oncogene 18:2681-89 (1999)). HB-EGF, β-cellulin and epiregulinalso bind to ErbB4.

[0011] While EGF and TGF do not bind ErbB2, EGF stimulates EGFR andErbB2 to form a heterodimer, which activates EGFR and results intransphosphorylation of ErbB2 in the heterodimer. Dimerization and/ortransphosphorylation appear to activate the ErbB2 tyrosine kinase. SeeEarp et al., supra. Likewise, when ErbB3 is co-expressed with ErbB2, anactive signaling complex is formed and antibodies directed against ErbB2are capable of disrupting this complex (Sliwkowski et al, J. Biol.Chem., 269(20):14661-14665 (1994)). Additionally, the affinity of ErbB3for heregulin (HRG) is increased to a higher affinity state whenco-expressed with ErbB2. See also, Levi et al., Journal of Neuroscience15: 1329-1340 (1995); Morrissey et al., Proc. Natl. Acad. Sci. USA 92:1431-1435 (1995); and Lewis et al., Cancer Res., 56:1457-1465 (1996)with respect to the ErbB2-ErbB3 protein complex. ErbB4, like ErbB3,forms an active signaling complex with ErbB2 (Carraway and Cantley, Cell78:5-8 (1994)).

[0012] Because of the physiological importance, members of the ErbBfamily of receptor tyrosine kinases have often been targeted fortherapeutic development. For example, Hudziak et al., Mol. Call. Biol.9(3):1165-1172 (1989) describe the generation of a panel of anti-ErbB2antibodies one of which, called 4D5, inhibited cellular proliferation by56%. A recombinant humanized version of the murine anti-ErbB2 antibody4D5 (huMAb4D5-8, rhuMAb HER2 or HERCEPTIN®; U.S. Pat. No. 5,821,337) isclinically active in patients with ErbB2-overexpressing metastaticbreast cancers that have received extensive prior anti-cancer therapy(Baselga et al., J. Clin. Oncol. 14:737-744 (1996)). HERCEPTIN® receivedmarketing approval from the Food and Drug Administration Sep. 25, 1998for the treatment of patients with metastatic breast cancer whose tumorsoverexpress the ErbB21HER2 protein. Since HER2 is also overexpressed inother cancers, in addition to breast cancer, HERCEPTIN® holds a greatpotential in the treatment of such other cancers as well.

[0013] 2. Smooth Muscle Cell Proliferation

[0014] Smooth muscle cells are very important structural and functionalcomponents of many hollow passages in the body, including blood vessels,gastrointestinal tract, airway passage (trachea and bronchi in lungs),urinary tract system (bladder and ureters) etc. They are responsible forelasticity that is so crucially required for normal functioning of theseorgans. They respond to a variety of physiological stimuli byconstriction or dilation as needed, for example, for regulating the flowof fluids carried by them. They respond not only to chemical stimuli,such as growth factors and cytokines, but also to physical stimuli, suchas pressure and stretch. Excessive proliferation of smooth muscle cellsresults in thickening of the wall and narrowing the lumen of the organsknown as “stenosis” in a variety of disorders.

[0015] A number of growth factors and cytokines are implicated in theproliferation of smooth muscle cells. One category of such importantmolecules are EGF related ligands. For example, smooth muscle cells froma variety of such organs have been demonstrated to possess EGFreceptors, and some of them even synthesize and secrete EGF ligands suchas HB-EGF, thus setting up autocrine loop. Various EGF ligands act aspotent mitogens and stimulate proliferation of smooth muscle cells oftenresulting in thickening of the wall and ultimately stenosis. Forexample, excessive proliferation of vascular smooth muscle cells (VSMC)is involved in pathology of vascular stenosis, restenosis resulting fromangioplasy or surgery or stent implants, atherosclerosis, transplantatherosclerosis and hypertension (reviewed in Casterella and Teirstein,Cardiol. Rev. 7: 219-231 [1999]; Andres, Int. J. Mol. Med. 2: 81-89[1998]; and Rosanio at al, Thromb. Haemost. 82 [suppl 1]: 164-170[1999]). The thickening of blood vessels increases resistance to bloodflow and ultimately leads to hypertension. Moreover, decreased bloodsupply to the tissue may also cause necrosis and induce inflammatoryresponse leading to severe damage. For example, myocardial infarctionoccurs as a result of lack of oxygen and local death of heart muscletissues.

[0016] Infantile hypertrophic pyloric stenosis (IHPS), which causesfunctional obstruction of the pyloric canal also involves hypertrophyand hyperplasia of the pyloric smooth muscle cells (Due and Puri,Pediatr. Res. 45: 853-857 [1999]). Furthermore, EGF, EGF receptor andHB-EGF are implicated in pathogenesis of pyloric stenosis (Shima et al.,Pediatr. Res. 47: 201-207 [2000]).

[0017] Similarly, the urinary bladder wall thickening that occurs inresponse to obstructive syndromes affecting the lower urinary tractinvolves proliferation of urinary bladder smooth muscle cells. Amembrane-bound precursor form of HB-EGF is expressed in urinary bladdersmooth muscle cells and HB-EGF is a potent mitogen for bladder SMCproliferation (Freeman et al., J. Clin. Invest. 99: 1028-1036 [1997];Kaefer et al., J. Urol. 163: 580-584 [2000]; Borer et al., Lab Invest.79: 1335-1345 [1999]).

[0018] The obstructive airway diseases are yet another group of diseaseswith underlying pathology involving smooth muscle cell proliferation.One example of this group is asthma which manifests in airwayinflammation and bronchoconstriction. EGF is implicated in thepathological proliferation of airway SMCs in obstructive airway diseases(Cerutis et al., Am. J. Physiol. 273: L10-15 [1997]; Cohen et al, Am. J.Respir. Cell. Mol. Biol. 16: 85-90 [1997]).

[0019] The instant invention discloses the use of ErbB4 receptorantagonists for controlling excessive migration and/or proliferation orsmooth muscle cells and, in particular, for the treatment of stenosis.

SUMMARY OF THE INVENTION

[0020] In one aspect, the invention concerns a method for controllingexcessive proliferation or migration of smooth muscle cells by treatingthe smooth muscle cells with an effective amount of an antagonist of anative ErbB4 receptor. The control is prevention or inhibition,including total inhibition, of excessive proliferation or migration ofsmooth muscle cells. In one embodiment the smooth muscle cells areurinary bladder smooth muscle cells, and in another embodiment they arethe smooth muscle cells of an airway passage.

[0021] The excessive proliferation or migration of smooth muscle cellssuch as vascular smooth muscle cells may result in stenosis includingvascular stenosis and restenosis. In one embodiment the smooth musclecells are human.

[0022] The stenosis may be further characterized by excessiveproliferation or migration of endothelial cells.

[0023] In one embodiment the ErbB4 receptor antagonist is animmunoadhesin. In another embodiment the ErbB4 receptor antagonist is anantibody, such as a neutralizing antibody against a native ErbB4receptor.

[0024] In another aspect, the invention concerns a method for treatingstenosis in a mammalian patient, including a human, comprisingadministering to the patient an effective amount of an antagonist of anative mammalian ErbB4 receptor. The treatment includes prevention ofstenosis. The stenosis may be vascular stenosis including restenosis.The antagonist may be administered as an injection or infusion. Thetreatment may also be used to reduce hypertension associated with thestenosis. The stenosis may be vascular stenosis including restenosis,pyloric stenosis, thickening of the urinary bladder wall or part of anobstructive airway disease.

[0025] In one embodiment the antagonist is an immunoadhesin, which maycomprise the extracellular region of a native human ErbB4 receptor. Inanother embodiment the antagonist is an antibody, such as a neutralizingantibody against a native human ErbB4 receptor.

[0026] In a further aspect, the invention concerns a method for treatingstenosis in a mammalian patient, such as a human, comprising introducinginto a cell of the patient a nucleic acid encoding an antagonist of anErbB4 receptor. The nucleic acid may be introduced in vivo or ex vivo,and with the aid of a vector such as retroviral vector or a lipid baseddelivery system. The method of the present invention is particularlyuseful for the treatment (including prevention) of vascular stenosis andrestenosis.

[0027] The antagonist may be an immunoadhesin. The antagonist may alsobe an antibody, such as a neutralizing antibody against a native humanErbB4 receptor.

[0028] In another aspect, the invention concern a method for treatinghypertension associated with vascular stenosis in a mammalian patient,comprising administering to the patient an effective amount of anantagonist of a native ErbB4 receptor. The antagonist may be a smallmolecule.

[0029] In a still further aspect, the invention concerns apharmaceutical composition for the treatment of stenosis in a mammalianpatient comprising an effective amount of an antagonist of a nativemammalian ErbB4 receptor, in admixture with a pharmaceuticallyacceptable carrier.

[0030] In all aspects, preferred ErbB4 antagonists includeimmunoadhesins, preferably comprising a native human ErbB4 receptorextracellular domain sequence fused to an immunoglobulin constant regionsequence. The immunoglobulin sequence preferably is that of a heavychain constant region of an IgG1, IgG2 or IgG3 immunoglobulin and mayadditionally comprise an immunoglobulin light chain sequence covalentlyattached to the fusion molecule comprising the immunoglobulin heavychain constant region.

[0031] Another preferred class of ErbB4 antagonists comprisesneutralizing antibodies specifically binding a native ErbB4 receptor.The antibodies preferably are human or humanized. In one embodiment theantibodies bind essentially the same epitope as an antibody produced bya hybridoma selected from the group consisting of HER4.01H1.1A1 (ATCCAccession Number PTA-2828), HER4.1C6.A11 (ATCC Accession NumberPTA-2829), HER4.3B9.2C9 (ATCC Accession Number PTA-2826), HER4.1A6.5B3(ATCC Accession Number PTA-2827) and HER4.8B1.2H2 (ATCC Accession NumberPTA-2825). The antibodies may also have complementarity determiningregion (CDR) residues from an antibody produced by a hybridoma selectedfrom the group consisting of HER4.10H1.1A1 (ATCC Accession NumberPTA-2828), HER4.1C6.A11 (ATCC Accession Number PTA-2829), HER4.3B9.2C9(ATCC Accession Number PTA-2826), HER4.1A6.5B3 (ATCC Accession NumberPTA-2827) and HER4.8B1.2H2 (ATCC Accession Number PTA-2825).

[0032] The smooth muscle cells may, for example, be pyloric or urinarybladder smooth muscle cells, or smooth muscle cells of an airwaypassage. Preferably, the smooth muscle cells are vascular smooth musclecells.

[0033] In a still further aspect, the invention concerns a method foridentifying a molecule that inhibits or enhances the proliferation ormigration of smooth muscle cells, comprising the steps of: (a)contacting a polypeptide comprising an amino acid sequence having atleast 85% sequence identity with the amino acid sequence of theextracellular domain of a native ErbB4 receptor and retaining theability to control excessive proliferation or migration of smooth musclecells, with a candidate molecule; and (b) determining whether thecandidate molecule inhibits or enhances the ability of the polypeptideto control excessive proliferation or migration of smooth muscle cells.The polypeptide may comprise the extracellular domain of a native ErbB4receptor. The polypeptide is an immunoadhesin in one embodiment. In aparticular embodiment, the molecule enhances the ability of thepolypeptide to control excessive proliferation or migration of smoothmuscle cells, and is an antibody or a small molecule.

[0034] In a yet further aspect the invention concerns an antibody thatbinds essentially the same epitope of ErbB4 as an antibody produced by ahybridoma selected from the group consisting of HER4.10H1.1A1 (ATCCAccession Number PTA-2828), HER4.1C6.A11 (ATCC Accession NumberPTA-2829), HER4.3B9.2C9 (ATCC Accession Number PTA-2826), HER4.1A6.5B3(ATCC Accession Number PTA-2827) and HER4.881.2H2 (ATCC Accession NumberPTA 2325). In addition to the methods set forth above and throughout thedisclosure, these antibodies are believed to be useful in the treatmentof various cancers, including breast cancer.

[0035] In a still further aspect the invention concerns an antibody thathas complementarity determining region (CDR) residues from an antibodyproduced by a hybridoma selected from the group consisting ofHER4.10H1.1A1 (ATCC Accession Number PTA-2828), HER4.1C6.A11 (ATCCAccession Number PTA-2829), HER4.3B9.2C9 (ATCC Accession NumberPTA-2826), HER4.1A5.5B3 (ATCC Accession Number PTA-2827) andHER4.8B1.2H2 (ATCC Accession Number PTA-2825).

[0036] In a further aspect the invention concerns an antibody selectedfrom the group consisting of an antibody produced by a hybridomaselected from the group consisting of HER4.10H1.1A1 (ATCC AccessionNumber PTA-2828), HER4.1C6.A11 (ATCC Accession Number PTA-2829),HER4.3B9.2C9 (ATCC Accession Number PTA-2826), HER4.1A6.5B3 (ATCCAccession Number PTA-2827) and HER4.8B1.2H2 (ATCC Accession NumberPTA-2825).

[0037] The invention also concerns an antibody that binds essentiallythe same epitope of ErbB4 bound by an antibody selected from the groupconsisting of anti-ErbB4 monoclonal antibodies 4-1440, 4-1460, 4-1473,4-1492 and 4-1464.

[0038] Further, the invention concerns an antibody that hascomplementarity determining region (CDR) residues from an antibodyselected from the group consisting of anti-ErbB4 monoclonal antibodies4-1440, 4-1460, 4-1473, 4-1492 and 4-1464.

[0039] The invention also concerns an antibody that binds ErbB4 withhigh affinity. This antibody preferably binds to ErbB4 with a Kd of lessthan 100 nM, more preferably with a Kd of less than 50 nM, even morepreferably with a Kd of less than 25 nM and most preferably with a Kdless than 10 nM. In one embodiment this antibody is a human antibody andin another embodiment it is a humanized antibody. In yet anotherembodiment the antibody is an antibody fragment.

[0040] The invention further concerns an antibody which is capable ofbinding to both ErbB4 and ErbB3. In one embodiment the antibody iscapable of binding ErbB4 with high affinity and in another embodimentthe antibody binds both ErbB4 and ErbB3 with high affinity.

[0041] In another aspect the invention concerns an antibody which bindsto ErbB4 and reduces heregulin binding thereto. This antibody may bindErbB4 with high affinity.

[0042] Finally, the invention concerns an antibody which binds to ErbB4and reduces heregulin-induced tyrosine phosphorylation thereof. Thisantibody may also bind ErbB4 with high affinity.

BRIEF DESCRIPTION OF THE DRAWINGS

[0043]FIG. 1 shows the nucleotide sequence of human ErbB4 (SEQ ID NO:1).

[0044]FIG. 2 shows the deduced amino acid sequence of human ErbB4 (SEQID NO: 2).

[0045]FIG. 3 shows the nucleotide sequence of an ErbB4-IgG immunoadhesin(SEQ ID NO: 3).

[0046]FIG. 4 shows the amino acid sequence of the ErbB4 extracellulardomain (ECD), which comprises amino acids 26 through 640 (SEQ ID NO: 4)of the ErbB4 amino acid sequence presented in FIG. 2 (SEQ ID NO: 2).

[0047]FIG. 5 shows the effect of ErbB4-IgG immunoadhesin onPDGF-stimulated proliferation of human aortic smooth muscle cells.

[0048]FIG. 6 shows the effect of ErbB4-IgG immunoadhesin on thechemotactic response of human aortic smooth muscle cells to thrombin.

[0049]FIG. 7 shows the inhibition of heregulin binding to HER4immunoadhesin by anti-HER4 monoclonal antibodies.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0050] A. Definitions

[0051] Unless defined otherwise, technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. See, e.g. Singleton etal., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley& Sons (New York, N.Y. 1994); Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, N.Y.1989). For purposes of the present invention, the following terms aredefined below.

[0052] Unless indicated otherwise, the term “ErbB” when used hereinrefers to any one or more of the mammalian ErbB receptors (i.e. ErbB1 orepidermal growth factor (EGF) receptor; ErbB2 or HER2 receptor; ErbB3 orHER3 receptor; ErbB4 or HER4 receptor; and any other member(s) of thisclass I tyrosine kinase family to be identified in the future) and“erbB” refers to the mammalian erbB genes encoding these receptors.

[0053] The terms “ErbB4” and “HER4” are used interchangeably and referto a native sequence ErbB4 receptor polypeptide as disclosed, forexample, in European Patent Application No. (EP) 599,274; Plowman atal., Proc. Natl. Acad. Sci. USA, 90:1746-1750 (1993); and Plowman etal., Nature, 366:473-475 (1993), and functional derivatives, includingamino acid sequence variants thereof.

[0054] A “native” or “native sequence” ErbB4 or HER4 receptor has theamino acid sequence of a naturally occurring ErbB4 receptor in anymammalian (including humans) species, irrespective of its mode ofpreparation. Accordingly, a native or native sequence ErbB4 receptor maybe isolated from nature, produced by techniques of recombinant DNAtechnology, chemically synthesized, or produced by any combinations ofthese or similar methods. Native ErbB4 receptors specifically includepolypeptides having the amino acid sequence of naturally occurringallelic variants, isoforms or spliced variants of ErbB4, known in theart or hereinafter discovered. Native sequence ErbB4 receptors aredisclosed, for example, in EP 599,274, supra, and in the two Plowman etal. papers, supra. Elenius et al., J. Biol. Chem. 272:26761-26768 (1997)report the identification of two alternatively spliced isoforms of ErbB4both in mouse and human tissues, that differ by the insertion of either23 (HER4 JM-a) or 13 (HER4 JM-b) alternative amino acids in theextracellular juxtamembrane (JM) region. Elenius et al., Oncogene18:2607-2615 (1999) report the identification and characterization ofanother naturally occurring isoform of ErbB4 (designated as ErbB4CYT-2), with a deletion of the cytoplasmic domain sequence required forthe activation of the PI3-K intracellular signal transduction pathway.HER4 isoforms are also disclosed in WO 99/19488. A nucleotide sequenceencoding ErbB4 is presented in FIG. 1 (SEQ ID NO: 1) and thecorresponding deduced amino acid sequence is depicted in FIG. 2 (SEQ IDNO: 2).

[0055] The term “ErbB4 extracellular domain” or “ErbB4 ECD” refers to asoluble fragment of ErbB4 comprising the amino acids located between thesignal sequence and the first predicted transmembrane region. In oneembodiment, the “ErbB4 ECD” is a polypeptide comprising amino acids26-640 (SEQ ID NO: 4) of the human ErbB4 sequence presented in FIG. 2(SEQ ID NO: 2).

[0056] The term “mammal” is used herein to refer to any animalclassified as a mammal, including, without limitation, humans, domesticand farm animals, and zoo, sports, or pet animals, such as sheep, dogs,horses, cats, cows, etc. Preferably, the mammal herein is human.

[0057] “Functional derivatives” include amino acid sequence variants,and covalent derivatives of the native polypeptides as long as theyretain a qualitative biological activity of the corresponding nativepolypeptide. Amino acid sequence variants generally differ from a nativesequence in the substitution, deletion and/or insertion of one or moreamino acids anywhere within a native amino acid sequence. Deletionalvariants include fragments of the native polypeptides, and variantshaving N- and/or C-terminal truncations. Ordinarily, amino acid sequencevariants will possess at least about 70% homology, preferably at leastabout 80%, more preferably at least about 90% homology with a nativepolypeptide.

[0058] “Homology” is defined as the percentage of residues in the aminoacid sequence variant that are identical after aligning the sequencesand introducing gaps, if necessary, to achieve the maximum percenthomology. Methods and computer programs for the alignment are well knownin the art. One such computer program is “Align 2”, authored byGenentech, Inc., which was filed with user documentation in the UnitedStates Copyright Office, Washington, D.C. 20559, on Dec. 10, 1991.

[0059] An ErbB “antagonist” is a molecule, which prevents or interfereswith an ErbB effector function, e.g. a molecule, which prevents orinterferes with binding and/or activation of a native sequence ErbBreceptor by a ligand, and/or downstream pathways used by the nativesequence ErbB receptor. Such molecules can be screened, for example,based upon their ability to competitively inhibit ErbB receptoractivation by ligand in the tyrosine phosphorylation assay. Similarly,an antagonist of a native sequence ErbB4 (HER4) receptor is a moleculewhich prevents or interferes with an ErbB4 effector function, e.g. amolecule which prevents or interferes with binding and/or activation ofa native sequence ErbB4 receptor by a ligand, and/or downstream pathwaysused by the ErbB4 receptor. Such molecules can be screened, for example,based upon their ability to competitively inhibit ErbB4 receptoractivation by ligand in the tyrosine phosphorylation assay. Examples ofErbB4 antagonists include, without limitation, soluble ErbB4 receptors(such as extracellular domains (ECD) of native sequence and variantErbB4 receptors), neutralizing antibodies against native sequence ErbB4receptors, neutralizing antibodies to ligands of native sequence ErbB4receptors (e.g. anti-HB-EGF antibodies), ErbB4-Ig immunoadhesins(including chimeric heteroadhesins) and small molecules.

[0060] By “ErbB4 ligand” is meant a polypeptide which binds to and/oractivates an ErbB4 receptor. ErbB4 ligands include betacellulin,epiregulin, HB-EGF, NRG-2, NRG-3 and heregulins.

[0061] In the methods of the present invention, the term “control” andgrammatical variants thereof, are used to refer to the prevention,partial or complete inhibition, reduction, delay or slowing down of anunwanted event, e.g. physiological condition, such as the excessiveproliferation and/or migration of smooth muscle cells and/or other celltypes, e.g. endothelial cells.

[0062] The term “excessive proliferation and/or migration” meansproliferation and/or migration beyond normal levels that results or islikely to result, if untreated, in the development of an unwantedphysiological condition or disease, such as, for example, stenosis,including vascular stenosis, restenosis, and pyloric stenosis; urinarybladder wall thickening, and obstructive airway disease.

[0063] “Treatment” refers to both therapeutic treatment and prophylacticor preventative measures. Those in need of treatment include thosealready with the disorder as well as those prone to have the disorder orthose in which the disorder is to be prevented. For purposes of thisinvention, beneficial or desired clinical results include, but are notlimited to, alleviation of symptoms, diminishment of extent of disease,stabilized (i.e., not worsening) state of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which thecondition or disorder is to be prevented.

[0064] The term “isolated” molecule is defined broadly as a moleculethat is identified and separated from at least one contaminant moleculewith which it is ordinarily associated in the natural source of themolecule. Preferably, the isolated molecule is free of association withall components with which it is naturally associated.

[0065] The term immunoadhesin” as used herein refers to antibody-likemolecules that combine the binding domain of a protein such as anextracellular domain (the adhesin portion) of a cell-surface receptorwith the effector functions of an immunoglobulin constant domain. Theterm “immunoadhesin” specifically includes native or variant ErbB4receptor sequences. The nucleic acid sequence of an ErbB4-IgGimmunoadhesin is presented in FIG. 3 (SEQ ID NO: 3). Immunoadhesins canpossess many of the valuable chemical and biological properties of humanantibodies. Since immunoadhesins can be constructed from a human proteinsequence with a desired specificity linked to an appropriate humanimmunoglobulin hinge and constant domain (Fc) sequence, the bindingspecificity of interest can be achieved using entirely human components.Such immunoadhesins are minimally immunogenic to the patient, and aresafe for chronic or repeated use. The term “isolated immunoadhesin”refers to an immunoadhesin that has been purified from a source or hasbeen prepared by recombinant or synthetic methods and is sufficientlyfree of other peptides or proteins.

[0066] Immunoadhesins reported in the literature include fusions of theT cell receptor (Gascoigne at al., Proc. Natl. Acad. Sci. USA84:2936-2940 (1987)1; CD4 (Capon et al., Nature 337:525-531 (1989);Traunecker et al., Nature 339:68-70 (1989); Zettmeissl et al., DNA CellBiol. USA 9:347-353 (1990); and Byrn et al., Nature 344:667-670 (1990));L-selectin or homing receptor (Watson et al., J. Cell. Biol.110:2221:2229 (1990); and Watson at al., Nature 349:164-167 (1991));CD44 (Aruffo at al., Cell 61:1303-1313 (1990)); CD28 and B7 (Linsley etal., J. Exp. Med. 173:721-730 (1991)); CTLA-4 (Lisley at al., J. Exp.Med. 174:561-569 (1991)); CD22 (Stamenkovic at al., Cell 66:1133-1144(1991)); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA88:10535-10539 (1991); Lesslauer et al., Eur. J. Immunol. 27:2883-2886(1991); and Peppel at al., J. Exp. Med. 174:1483-1489 (1991)); NPreceptors (Bennett et al., J. Biol. Chem. 266:23060-23067 (1991));inteferon receptor (Kurschner et al., J. Biol. Chem. 267:9354-9360(1992)); 4-1BB (Chalupny et al., PNAS USA 89:10360-10364 (1992)) and IgEreceptor (Ridgway and Gorman, J. Cell. Biol. 115, Abstract No. 1448(1991)).

[0067] Examples of homomultimeric immunoadhesins which have beendescribed for therapeutic use include the CD4-IgG immunoadhesin forblocking the binding of HIV to cell-surface CD4. Data obtained fromPhase I clinical trials, in which CD4-IgG was administered to pregnantwomen just before delivery, suggests that this immunoadhesin may beuseful in the prevention of maternal-fetal transfer of HIV (Ashkenazi etal., Intern. Rev. Immunol. 10:219-227 (1993). An immunoadhesin whichbinds tumor necrosis factor (TNF) has also been developed. TNF is aproinflammatory cytokine which has been shown to be a major mediator ofseptic shock. Based on a mouse model of septic shock, a TNF receptorimmunoadhesin has shown promise as a candidate for clinical use intreating septic shock (Ashkenazi, A. et al. (1991) PNAS USA88:10535-10539). ENBREL® (etanercept), an immunoadhesin comprising a TNFreceptor sequence fused to an IgG Fc region, was approved by the U.S.Food and Drug Administration (FDA), on Nov. 2, 1998, for the treatmentof rheumatoid arthritis. The new expanded use of ENBREL® in thetreatment of rheumatoid arthritis has recently been approved by FDA onJun. 6, 2000. For recent information on TNF blockers, including ENBREL®,see Lovell et al., N. Engl. J. Med. 342: 763-169 (2000), andaccompanying editorial on p810-811; and Weinblatt et al., N. Engl. J.Med. 340: 253-259 (1999); reviewed in Maini and Taylor, Annu. Rev. Med.51: 207-229 (2000). Immunoadhesins also have non-therapeutic uses. Forexample, the L-selectin receptor immunoadhesin was used as a reagent forhistochemical staining of peripheral lymph node high endothelial venules(HEV). This reagent was also used to isolate and characterize theL-selectin ligand (Ashkenazi at al., supra).

[0068] If the two arms of the immunoadhesin structure have differentspecificities, the immunoadhesin is called a “bispecific immunoadhesin”by analogy to bispecific antibodies. Dietsch et al., J. Immunol. Methods162:123 (1993) describe such a bispecific immunoadhesin combining theextracellular domains of the adhesion molecules, E-selectin andP-selectin, each of which selectins is expressed in a different celltype in nature. Binding studies indicated that the bispecificimmunoglobulin fusion protein so formed had an enhanced ability to bindto a myeloid cell line compared to the monospecific immunoadhesins fromwhich it was derived.

[0069] The term “heteroadhesin” is used interchangeably with theexpression “chimeric heteromultimer adhesin” and refers to a complex ofchimeric molecules (amino acid sequences) in which each chimericmolecule combines a biologically active portion, such as theextracellular domain of each of the heteromultimeric receptor monomers,with a multimerization domain. The “multimerization domain” promotesstable interaction of the chimeric molecules within the heteromultimercomplex. The multimerization domains may interact via an immunoglobulinsequence, leucine zipper, a hydrophobic region, a hydrophilic region, ora free thiol which forms an intermolecular disulfide bond between thechimeric molecules of the chimeric heteromultimer. The multimerizationdomain may comprise an immunoglobulin constant region. In addition amultimerization region may be engineered such that steric interactionsnot only promote stable interaction, but further promote the formationof heterodimers over homodimers from a mixture of monomers.“Protuberances” are constructed by replacing small amino acid sidechains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory ‘cavities’ ofidentical or similar size to the protuberances are optionally created onthe interface of the second polypeptide by replacing large amino acidside chains with smaller ones (e.g. alanine or threonine). Theimmunoglobulin sequence preferably, but not necessarily, is animmunoglobulin constant domain. The immunoglobulin moiety in thechimeras of the present invention may be obtained from IgG₁, IgG₂, IgG₃or IgG₄ subtypes, IgA, IgE, IgD or IgM, but preferably IgG₁ or IgG₃.

[0070] The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising the entire chimeric heteroadhesin, or a fragmentthereof, fused to a “tag polypeptide”. The tag polypeptide has enoughresidues to provide an epitope against which an antibody can be made,yet is short enough such that it does not interfere with activity of thechimeric heteroadhesin. The tag polypeptide preferably is fairly uniqueso that the antibody thereagainst does not substantially cross-reactwith other epitopes. Suitable tag polypeptides generally have at least 6amino acid residues and usually between about 8-50 amino acid residues(preferably between about 9-30 residues). An embodiment of the inventionencompasses a chimeric heteroadhesin linked to an epitope tag, which tagis used to detect the adhesin in a sample or recover the adhesin from asample.

[0071] “Isolated/highly purified/substantially homogenousimmunoadhesin”, “isolated/highly purified/substantially homogenousheteroadhesin”, and “isolated/highly purified/substantially homogenouschimeric heteromultimer adhesin”, are used interchangeably and mean theadhesin that has been purified from a source or has been prepared byrecombinant or synthetic methods and is sufficiently free of otherpeptides or proteins to homogeneity by chromatographic techniques orother purification techniques, such as SDS-PAGE under nonreducing orreducing conditions using Coomassie blue or, preferably, silver stain.Homogeneity here means less than about 5% contamination with othersource proteins. The ErbB214-IgG chimeric heteroadhesins of theinvention bind with sufficiently greater affinity relative to thehomodimers that the use of a mixture of homodimers and heterodimers isalso considered a useful embodiment of the invention. The terms“chimeric heteromultimer adhesin”, “chimeric heteroadhesin” and “CHA”are used interchangeably herein.

[0072] The term “antibody” is used in the broadest sense andspecifically covers antibodies that recognize native ErbB4 receptors. Anantibody that shows “high affinity” binding has a Kd of less than about100 nM, preferably less than about 50, more preferably less than about25, most preferably less than about 10.

[0073] The term “monoclonal antibody” as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally-occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigenic site. Furthermore, in contrastto conventional (polyclonal) antibody preparations which typicallyinclude different antibodies directed against different determinants(epitopes), each monoclonal antibody is directed against a singledeterminant on the antigen.

[0074] The monoclonal antibodies herein include hybrid and recombinantantibodies produced by splicing a variable (including hypervariable)domain of an anti-chimeric heteroadhesin antibody with a constant domain(e.g. “humanized” antibodies), or a light chain with a heavy chain, or achain from one species with a chain from another species, or fusionswith heterologous proteins, regardless of species of origin orimmunoglobulin class or subclass designation, as well as antibodyfragments (e.g., Fab, F(ab)₂, and Fv), so long as they exhibit thedesired biological activity. (See, e.g., U.S. Pat. No. 4,816,567 andMage & Lamoyi, in Monoclonal Antibody Production Techniques andApplications, pp.79-97 (Marcel Dekker, Inc.), New York (1987)).

[0075] Thus, the modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler & Milstein,Nature 256:495 (1975), or may be made by recombinant DNA methods (U.S.Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolatedfrom phage libraries generated using the techniques described inMcCafferty et al, Nature 348:552-554 (1990), for example.

[0076] “Humanized” forms of non-human (e.g. murine) antibodies arespecific chimeric immunoglobulins, immunoglobulin chains or fragmentsthereof (such as Fv, Fab, Fab′, F(ab)₂ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. For the most part, humanized antibodies arehuman immunoglobulins (recipient antibody) in which residues from thecomplementarity determining regions (CDRs) of the recipient antibody arereplaced by residues from the CDRs of a non-human species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human FR residues. Furthermore, the humanized antibody may compriseresidues which are found neither in the recipient antibody nor in theimported CDR or FR sequences. These modifications are made to furtherrefine and optimize antibody performance. In general, the humanizedantibody will comprise substantially all of at least one, and typicallytwo, variable domains, in which all or substantially all of the CDRregions correspond to those of a non-human immunoglobulin and all orsubstantially all of the FR residues are those of a human immunoglobulinconsensus sequence. The humanized antibody optimally also will compriseat least a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin.

[0077] “Muscle cells” include skeletal, cardiac or smooth muscle tissuecells. This term encompasses those cells which differentiate to formmore specialized muscle cells (e.g. myoblasts). Vascular smooth musclecells refer to smooth muscle cells present in a middle elastic layer,media, of blood vessels.

[0078] The term “stenosis” refers to narrowing or stricture of a hollowpassage (e,g, a duct or canal) in the body. The term “vascular stenosis”refers to occlusion or narrowing of blood vessels. Vascular stenosisoften results from fatty deposit (as in the case of atherosclerosis) orexcessive migration and proliferation of vascular smooth muscle cellsand endothelial cells. Arteries are particularly susceptible tostenosis. The term “stenosis” as used herein specifically includesinitial stenosis and restenosis.

[0079] The term “restenosis” refers to recurrence of stenosis aftertreatment of initial stenosis with apparent success. For example,“restenosis” in the context of vascular stenosis, refers to thereoccurrence of vascular stenosis after it has been treated withapparent success, e.g. by removal of fatty deposit by balloonangioplasty. One of the contributing factors in restenosis is intimalhyperplasia. The term “intimal hyperplasia”, used interchangeably with“neointimal hyperplasia” and “neointima formation”, refers to thickeningof the inner most layer of blood vessels, intima, as a consequence ofexcessive proliferation and migration of vascular smooth muscle cellsand endothelial cells. The various changes taking place duringrestenosis are often collectively referred to as “vascular wallremodeling.”

[0080] The terms “balloon angioplasty” and “percutaneous transluminalcoronary angioplasty” (PTCA) are often used interchangeably, and referto a non-surgical catheter-based treatment for removal of plaque fromthe coronary artery. Stenosis or restenosis often lead to hypertensionas a result of increased resistance to blood flow.

[0081] The term “pyloric stenosis” refers to narrowing of pylorus, thepassage at the lower end of the stomach that opens into the duodenum.

[0082] The term “hypertension” refers to abnormally high blood pressure,i.e. beyond the upper value of the normal range.

[0083] By “neutralizing antibody” is meant an antibody molecule asherein defined which is able to block or significantly reduce aneffector function of ErbB receptors. Accordingly, a “neutralizing”anti-ErbB4 antibody is capable of blocking or significantly reducing aneffector function, such as ligand binding and/or elicitation of acellular response, of ErbB4. For the purpose of the present invention,the ability of an anti-ErbB4 antibody to neutralize the binding of anErbB4 ligand (heregulin, HRG) to ErbB4 can be monitored, for example, bymeasuring the binding of detectably labeled HRG to purified ErbB4 or toa cell line exressing or modified to express ErbB4 in the presence andabsence of a candidate anti-ErbB4 antibody. Such assays are described inExample 4 below. For the purpose of the present invention, the abilityof the anti-ErbB4 antibodies to neutralize the elicitation of a cellularresponse by ErbB4 is preferably tested by monitoring the inhibition oftyrosine phosphorylation of ErbB4 by heregulin (HRG), or in a cellproliferation assay. Representative assays are disclosed in Example 4below. “Significant” reduction means at least about 60%, or at leastabout 70%, preferably at least about 75%, more preferably at least about80%, even more preferably at least about 85%, still more preferably atleast about 90%, still more preferably at least about 95%, mostpreferably at least about 99% reduction of an effector function of thetarget antigen (e.g. ErbB4), such as ligand (e.g. HRG) binding and/orelicitation of a cellular response. Preferably, the “neutralizing”antibodies as defined herein will be capable of neutralizing at leastabout 60%, or at least about 70%, preferably at least about 75%, morepreferably at least about 80%; even more preferably at least about 85%,still more preferably at least about 90%, still more preferably at leastabout 95%, most preferably at least about 99% of the tyrosinephosphorylation of ErbB4 by HRG, as determined by the assay described inExample 4.

[0084] An “isolated” antibody is one that has been identified andseparated and/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the antibody willbe purified (1) to greater than 95% by weight of antibody as determinedby the Lowry method, and most preferably more than 99% by weight, (2) toa degree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SOS-PAGE under reducing or non-reducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

[0085] The term “epitope” is used to refer to binding sites for(monoclonal or polyclonal) antibodies on protein antigens.

[0086] Antibodies which bind to a particular epitope can be identifiedby “epitope mapping.” There are many methods known in the art formapping and characterizing the location of epitopes on proteins,including solving the crystal structure of an antibody-antigen complex,competition assays, gene fragment expression assays, and syntheticpeptide-based assays, as described, for example, in Chapter 11 of Harlowand Lane, Using Antibodies, a Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1999. Competition assays arediscussed below. According to the gene fragment expression assays, theopen reading frame encoding the protein is fragmented either randomly orby specific genetic constructions and the reactivity of the expressedfragments of the protein with the antibody to be tested is determined.The gene fragments may, for example, be produced by PCR and thentranscribed and translated into protein in vitro, in the presence ofradioactive amino acids. The binding of the antibody to theradioactively labeled protein fragments is then determined byimmunoprecipitation and gel electrophoresis. Certain epitopes can alsobe identified by using large libraries of random peptide sequencesdisplayed on the surface of phage particles (phage libraries).Alternatively, a defined library of overlapping peptide fragments can betested for binding to the test antibody in simple binding assays. Thelatter approach is suitable to define linear epitopes of about 5 to 15amino acids.

[0087] An antibody binds “essentially the same epitope” as a referenceantibody, when the two antibodies recognize identical or stericallyoverlapping epitopes. The most widely used and rapid methods fordetermining whether two epitopes bind to identical or stericallyoverlapping epitopes are competition assays, which can be configured inall number of different formats, using either labeled antigen or labeledantibody. Usually, the antigen is immobilized on a 96-well plate, andthe ability of unlabeled antibodies to block the binding of labeledantibodies is measured using radioactive or enzyme labels.

[0088] The phrase “inhibiting an ErbB4 (HER4) receptor” refers to theability of an ErbB4 antagonist to inhibit or prevent activation of anErbB4 receptor, for example, by blocking the binding of a ligand to theErbB4 receptor. The “activation” of an ErbB4 receptor refers to receptorphosphorylation, which can be quantified using the tyrosinephosphorylation assays, and downstream events that constitute inductionof signal transduction by the bound ligand. “Inhibition” is any of theseassays is at least about 60%, or at least about 70%, preferably at leastabout 75%, more preferably at least about 80%; even more preferably atleast about 85%, still more preferably at least about 90%, still morepreferably at least about 95%, most preferably at least about 99%.

[0089] The expression “decreasing survival of a cell” refers to the actof decreasing the period of existence of a cell, relative to anuntreated cell which has not been exposed to a ErbB4 antagonist eitherin vitro or in vivo. The expression “decreased cell proliferation”refers to a decrease in the number of cells in a population exposed toan ErbB4 antagonist either in vitro or in vivo, relative to an untreatedcell.

[0090] “Biological activity” where used in conjunction with an ErbB4antagonist refers to the ability of an ErbB4 antagonist to control theexcessive proliferation or migration of smooth muscle cells, asdetermined in a relevant in vitro or in vivo assay, including thePDGF-stimulated smooth muscle cell proliferation and human aortic smoothmuscle cell migration assays described in the Examples hereinbelow,animal models and human clinical trials, irrespective of the underlyingmechanism. Thus, the biological activity of an ErbB4 antagonistincludes, without limitation, functioning as an inhibitor of the bindingof a ligand or activation of a native ErbB4 receptor, and/or inhibitionof growth and/or migration of smooth muscle cells expressing an ErbB4receptor on their surface.

[0091] The term “disease state” refers to a physiological state of acell or of a whole mammal in which an interruption, cessation, ordisorder of cellular or body functions systems, or organs has occurred.

[0092] The term “effective amount” refers to an amount of a drugeffective to treat (including prevention) a disease, disorder orunwanted physiological conditions in a mammal. In the present invention,an “effective amount” of an ErbB4 antagonist may reduce, slow down ordelay the proliferation of smooth muscle cells; reduce, slow down ordelay the migration of smooth muscle cells; prevent or inhibit (i.e.,slow to some extent and preferably stop) the development of stenosis orrestenosis; and/or relieve to some extent one or more of the symptomsassociated with stenosis or restenosis, in particular, prevent orinhibit (e., slow to some extent and preferably stop) the development ofelevated blood pressure associated with stenosis or restenosis.

[0093] Nucleic acid is “operably linked” when it is placed into afunctional relationship with another nucleic acid sequence. For example,DNA for a presequence or secretory leader is operably linked to DNA fora polypeptide if it is expressed as a preprotein that participates inthe secretion of the polypeptide; a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thesequence; or a ribosome binding site is operably linked to a codingsequence if it is positioned so as to facilitate translation. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inreading phase. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites. It suchsites do not exist, the synthetic oligonucleotide adapters or linkersare used in accordance with conventional practice.

[0094] “Pharmaceutically acceptable” carriers, excipients, orstabilizers are ones which are nontoxic to the cell or mammal beingexposed thereto at the dosages and concentrations employed. Often thephysiologically acceptable carrier is an aqueous pH buffered solution.Examples of physiologically acceptable carriers include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid; low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as Tween, polyethylene glycol(PEG), and Pluronics.

[0095] B. Methods for Carrying Out the Invention

[0096] The invention concerns the treatment of stenosis by antagonistsof native ErbB4 receptors. Although the invention is not so limited, ina preferred embodiment, the antagonist is an immunoadhesin or a chimericheteromultimer adhesin. Immunoadhesins (referred to as hybridimmunoglobulins), including their structure and preparation, aredescribed, e.g. in WO 91/08298; and in U.S. Pat. Nos. 5,428,130 and5,116,964, the disclosures of which are hereby expressly incorporated byreference.

[0097] 1. Production of an Immunoadhesin or Chimeric HeteromultimerAdhesin.

[0098] The description below relates primarily to production ofimmunoadhesin by culturing cells transformed or transfected with avector containing immunoadhesin nucleic acid. It is, of course,contemplated that alternative methods, which are well known in the art,may be employed to prepare immunoadhesin. For instance, theimmunoadhesin sequence, or portions thereof, may be produced by directpeptide synthesis using solid-phase techniques [see, e.g., Stewart etal., Solid-Phase Peptide Synthesis, W. H. Freeman Co., San Francisco,Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)]. Invitro protein synthesis may be performed using manual techniques or byautomation. Automated synthesis may be accomplished, for instance, usingan Applied Biosystems Peptide Synthesizer (Foster City, Calif.) usingmanufacturer's instructions. Various portions of the immunoadhesin maybe chemically synthesized separately and combined using chemical orenzymatic methods to produce the full-length immunoadhesin.

[0099] Nucleic acid encoding a native sequence ErbB4 receptor can, forexample, be isolated from cells known to express the ErbB4 receptor,such as those described in EP 599,274, supra, and in the collectivePlowman et al. references, supra or is synthesized.

[0100] DNA encoding immunoglobulin light or heavy chain constant regionsis known or readily available from cDNA libraries or is synthesized. Seefor example, Adams et al., Biochemistry 19:2711-2719 (1980); Gough etal., Biochemistry 19:2702-2710 (1980); Dolby et al; P.N.A.S. USA,77:6027-6031 (1980); Rice et al P.N.A.S USA 79:7862-7865 (1982); Falkneret al; Nature 298:286-288 (1982); and Morrison et al; Ann. Rev. Immunol.2:239-256 (1984).

[0101] An immunoadhesin or a chimeric heteroadhesin of the invention ispreferably produced by expression in a host cell and isolated therefrom.A host cell is generally transformed with the nucleic acid of theinvention. Preferably the nucleic acid is incorporated into anexpression vector. Suitable host cells for cloning or expressing thevectors herein are prokaryote host cells (such as E. coli, strains ofBacillus, Pseudomonas and other bacteria), yeast and other eukaryoticmicrobes, and higher eukaryote cells (such as Chinese hamster ovary(CHO) cells and other mammalian cells). The cells may also be present inlive animals (for example, in cows, goats or sheep). Insect cells mayalso be used. Cloning and expression methodologies are well known in theart.

[0102] To obtain expression of an immunoadhesin such as a chimericErbB4-IgG molecule, one or more expression vector(s) is/are introducedinto host cells by transformation or transfection and the resultingrecombinant host cells are cultured in conventional nutrient media,modified as appropriate for inducing promoters, selecting recombinantcells, or amplifying the ErbB4-IgG DNA. In general, principles,protocols, and practical techniques for maximizing the productivity ofin vitro mammalian cell cultures can be found in Mammalian CellBiotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991).

[0103] (i) Construction of Nucleic Acid Encoding Immunoadhesin

[0104] When preparing the immunoadhesins of the present invention,preferably nucleic acid encoding an extracellular domain of a naturalreceptor is fused C-terminally to nucleic acid encoding the N-terminusof an immunoglobulin constant domain sequence, however N-terminalfusions are also possible. Typically, in such fusions the encodedchimeric polypeptide will retain at least functionally active hinge, CH2and CH3 domains of the constant region of an immunoglobulin heavy chain.Fusions are also made to the C-terminus of the Fc portion of a constantdomain, or immediately N-terminal to the CH1 of the heavy chain or thecorresponding region of the light chain. The resultant DNA fusionconstruct is expressed in appropriate host cells.

[0105] Nucleic acid molecules encoding amino acid sequence variants ofnative sequence extracellular domains (such as from ErbB4) and/or theantibody sequences used to prepare the desired immunoadhesin, areprepared by a variety of methods known in the art. These methodsinclude, but are not limited to, isolation from a natural source (in thecase of naturally occurring amino acid sequence variants, such as thosementioned above in connection with ErbB4) or preparation byoligonucleotide-mediated (or site-directed) mutagenesis, PCRmutagenesis, and cassette mutagenesis of an earlier prepared variant ora non-variant version of native sequence ErbB4.

[0106] Amino acid sequence variants of native sequence extracellulardomain included in the chimeric heteroadhesin are prepared byintroducing appropriate nucleotide changes into the native extracellulardomain DNA sequence, or by in vitro synthesis of the desired chimericheteroadhesin monomer polypeptide. Such variants include, for example,deletions from, or insertions or substitutions of, residues in the aminoacid sequence of the immunoadhesin or chimeric heteroadhesin.

[0107] Variations in the native sequence as described above can be madeusing any of the techniques and guidelines for conservative andnon-conservative mutations set forth in Table 1.

[0108] In a preferred embodiment, the nucleic acid encodes a chimericmolecule in which the ErbB4 receptor extracellular domain sequence isfused to the N-terminus of the C-terminal portion of an antibody (inparticular the Fc domain), containing the effector functions of animmunoglobulin, e.g. IgG1. It is possible to fuse the entire heavy chainconstant region to the ErbB4 receptor extracellular domain sequence.However; more preferably, a sequence beginning in the hinge region justupstream of the papain cleavage site (which defines IgG Fc chemically;residue 216, taking the first residue of heavy chain constant region tobe 114 [Kobet et al., supra], or analogous sites of otherimmunoglobulins) is used in the fusion. In a particularly preferredembodiment, the ErbB4 receptor extracellular domain sequence is fused tothe hinge region and CH2 and CH3 or CH1, hinge, CH2 and CH3 domains ofan IgG1, IgG2, or IgG3 heavy chain. The precise site at which the fusionis made is not critical, and the optimal site can be determined byroutine experimentation.

[0109] For human immunoadhesins, the use of human IgG1 and IgG3immunoglobulin sequences is preferred. A major advantage of using IgG1is that IgG1 immunoadhesins can be purified efficiently on immobilizedprotein A. In contrast, purification of IgG3 requires protein G, asignificantly less versatile medium. However, other structural andfunctional properties of immunoglobulins should be considered whenchoosing the Ig fusion partner for a particular immunoadhesinconstruction. For example, the IgG3 hinge is longer and more flexible,so it can accommodate larger “adhesin” domains that may not fold orfunction properly when fused to IgG1. Another consideration may bevalency; IgG immunoadhesins are bivalent homodimers, whereas Ig subtypeslike IgA and IgM may give rise to dimeric or pentameric structures,respectively, of the basic Ig homodimer unit.

[0110] For ErbB4-Ig immunoadhesins designed for in vivo application, thepharmacokinetic properties and the effector functions specified by theFc region are important as well. Although IgG1, IgG2 and IgG4 all havein vivo half-lives of 21 days, their relative potencies at activatingthe complement system are different. IgG4 does not activate complement,and IgG2 is significantly weaker at complement activation than IgG1.Moreover, unlike IgG1, IgG2 does not bind to Fc receptors on mononuclearcells or neutrophils. While IgG3 is optimal for complement activation,its in viva half-life in approximately one third of the other IgGisotypes.

[0111] Another important consideration for immunoadhesins designed to beused as human therapeutics is the number of allotypic variants of theparticular isotype. In general, IgG isotypes with fewerserologically-defined allotypes are preferred. For example, IgG1 hasonly four serologically-defined allotypic sites, two of which (G1m and2) are located in the Fc region; and one of these sites G1m1, isnon-immunogenic. In contrast, there are 12 serologically-definedallotypes in IgG3, all of which are in the Fc region; only three ofthese sites (G3 m5, 11 and 21) have one allotype which isnonimmunogenic. Thus, the potential immunogenicity of an IgG3immunoadhesin is greater than that of an IgG1 immunoadhesin.

[0112] The cDNAs encoding the ErbB4 receptor sequence (e.g. anextracellular domain sequence) and the Ig parts of the immunoadhesin areinserted in tandem into a plasmid vector that directs efficientexpression in the chosen host cells. For expression in mammalian cellspRK5-based vectors [Schall et al., Cell 61, 361-370 (1990)] andCDM8-based vectors [Seed, Nature 329, 840 (1989)] may, for example, beused. The exact junction can be created by removing the extra sequencesbetween the designed junction codons using of oligonucleotide-directeddeletional mutagenesis [Zoller and Smith, Nucleic Acids Res. 10, 6487(1982); Capon et al., Nature 337, 525-531 (1989)]. Syntheticoligonucleotides can be used, in which each half is complementary to thesequence on either side of the desired junction; ideally, these are 36to 48-mers. Alternatively, PCR techniques can be used to join the twoparts of the molecule in-frame with an appropriate vector.

[0113] Although the presence of an immunoglobulin light chain is notrequired in the immunoadhesins of the present invention, animmunoglobulin light chain might be present either covalently associatedto an trk receptor-immunoglobulin heavy chain fusion polypeptide, ordirectly fused to the trk receptor extracellular domain. In the formercase, DNA encoding an immunoglobulin light chain is typicallycoexpressed with the DNA encoding the ErbB4 receptor-immunoglobulinheavy chain fusion protein. Upon secretion, the hybrid heavy chain andthe light chain will be covalently associated to provide animmunoglobulin-like structure comprising two disulfide-linkedimmunoglobulin heavy chain-light chain pairs. Method suitable for thepreparation of such structures are, for example, disclosed in U.S. Pat.No. 4,816,567 issued Mar. 28, 1989.

[0114] Another preferred type of chimeric ErbB4 antagonist herein is afusion protein comprising an extracellular domain, such as from a ErbB4monomer, linked to a heterologous polypeptide, such as a multimerizationdomain. Such a sequence can be constructed using recombinant DNAtechniques. Alternatively, the heterologous polypeptide can becovalently bound to the extracellular domain polypeptide by techniqueswell known in-the art such as the use of the heterobifunctionalcrosslinking reagents. Exemplary coupling agents includeN-succinimidyl-3-(2-pyridyidithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCLC, active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene).

[0115] In one embodiment, a chimeric heteroadhesin polypeptide comprisesa fusion of a monomer of the chimeric heteroadhesin with a tagpolypeptide which provides an epitope to which an anti-tag antibody canselectively bind. Such epitope tagged forms of the chimericheteroadhesin are useful, as the presence thereof can be detected usinga labeled antibody against the tag polypeptide. Also, provision of theepitope tag enables the chimeric heteroadhesin to be readily purified byaffinity purification using the anti-tag antibody. Tag polypeptides andtheir respective antibodies are well known in the art. Examples includethe flu HA tag polypeptide and its antibody 12CA5, (Field et al., Mol.Cell. Biol. 8:2159-2165 (1988)); the c-myc tag and the 8F9, 3C7, 6E10,G4, B7 and 9E10 antibodies thereto (Evan et al., Molecular and CellularBiology 5(12):3610-3616 (1985)); and the Herpes Simplex virusglycoprotein D (gD) tag and its antibody (Paborsky et al., ProteinEngineering 3(6):547-553 (1990)).

[0116] Another type of covalent modification of a chimericheteromultimer comprises linking a monomer polypeptide of theheteromultimer to one of a variety of non-proteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol, polyoxyalkylenes, orcopolymers of polyethylene glycol and polypropylene glycol. A chimericheteromultimer also may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization(for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively), in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, Oslo, A., Ed., (1980).

[0117] (ii) Selection and Transformation of Host Cells

[0118] Host cells are transfected or transformed with expression orcloning vectors described herein for immunoadhesin production andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences. The culture conditions, such as media,temperature, pH and the like, can be selected by the skilled artisanwithout undue experimentation. In general, principles, protocols, andpractical techniques for maximizing the productivity of cell culturescan be found in Mammalian Cell Biotechnology: a Practical Approach, M.Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.

[0119] The terms “transformation” and “transiection” are usedinterchangeably herein and refer to the process of introducing DNA intoa cell. Following transformation or transfection, the nucleic acid ofthe invention may integrate into the host cell genome, or may exist asan extrachromosomal element. Methods of eukaryotic cell transfection andprokaryotic cell transformation are known to the ordinarily skilledartisan, for example, CaCl₂, CaPO₄, liposome-mediated andelectroporation. Depending on the host cell used, transformation isperformed using standard techniques appropriate to such cells. Thecalcium treatment employing calcium chloride, as described in Sambrooket al., supra, or electroporation is generally used for prokaryotes.Infection with Agrobacterium tumefaciens is used for transformation ofcertain plant cells, as described by Shaw et al., Gene, 23:315 (1983)and WO 89/05859 published Jun. 29, 1989. For mammalian cells withoutsuch cell walls, the calcium phosphate precipitation method of Grahamand van der Eb, Virology, 52:456-457 (1978) can be employed. Generalaspects of mammalian cell host system transfections have been describedin U.S. Pat. No. 4,399,216. Transformations into yeast are typicallycarried out according to the method of Van Solingen et al., J. Bact.,130:946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829(1979). However, other methods for introducing DNA into cells, such asby nuclear microinjection, electroporation, bacterial protoplast fusionwith intact cells, or polycations, e.g., polybrene, polyornithine, mayalso be used. For various techniques for transforming mammalian cells,see Keown et al., Methods in Enzymology, 185:527-537 (1990) and Mansouret al., Nature, 336:348-352 (1988).

[0120] Suitable host cells for cloning or expressing the DNA in thevectors herein include prokaryote, yeast, or higher eukaryote cells.Suitable prokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting. Strain W3110 is one particularly preferred host or parenthost because it is a common host strain for recombinant DNA productfermentation. Preferably, the host cell secretes minimal amounts ofproteolytic enzymes. For example, strain W3110 may be modified to effecta genetic mutation in the genes encoding proteins endogenous to thehost, with examples of such hosts including E. coli W3110 strain 1A2,which has the complete genotype tonA; E. coli W3110 strain 9E4, whichhas the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC55,244), which has the complete genotype tonA ptr3 phoA E15(argF-lac)169 degP ompT kan′; E. coli W3110 strain 37D6, which has thecomplete genotype tonA ptr3phoA E15 (argF-lac)169 degP ompT rbs 7 ilvGkan′; E. coli W3110 strain 40B4, which is strain 3706 with anon-kanamycin resistant degP deletion mutation; and an E. coli strainhaving mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783issued Aug. 7, 1990. Alternatively, in vitro methods of cloning, e.g.,PCR or other nucleic acid polymerase reactions, are suitable.

[0121] In addition to prokaryotes, eukaryotic microbes such asfilamentous fungi or yeast are suitable cloning or expression hosts forimmunoadhesin-encoding vectors. Saccharomyces cerevisiae is a commonlyused lower eukaryotic host microorganism. Others includeSchizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 [1981]; EP139,383 published May 2, 1985); Kluyveromyces hosts (U.S. Pat. No.4,943,529; Fleer et al., Bio/Technology, 9:968-975 (1991)) such as,e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J.Bacteriol., 154(2): 737-1742 [1983]), K. fragilis (ATCC 12,424), K.bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al.,Bio/Technology, 8:135 (1990)), K. thermotolerans, and K. marxianus;yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al.,J. Basic Microbiol., 28:265-278 [1998)); Candida; Trichoderma reesia (EP244,234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA,76:5259-5263 [1979]); Schwanniomyces such as Schwanniomyces occidentalis(EP 394,538 published Oct. 31, 1990); and filamentous fungi such as,e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published Jan.10, 1991), and Aspergillus hosts such as A. nidulans (Ballance et al.,Biochem. Biophys. Res. Commun., 112:284-289 [1983]; Tilburn et al.,Gene, 26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479[1985]). Methylotropic yeasts are suitable herein and include, but arenot limited to, yeast capable of growth on methanol selected from thegenera consisting of Hansenula, Candida, Kloeckera, Pichia,Saccharomyces, Torulopsis, and Rhodotorula. A list of specific speciesthat are exemplary of this class of yeasts may be found in C. Anthony,The Biochemistry of Methylotrophs, 269 (1982).

[0122] Suitable host cells for the expression of glycosylatedimmunoadhesin are derived from multicellular organisms. Examples ofinvertebrate cells include insect cells such as Drosophila S2 andSpodoptera Sf9, as well as plant cells. Examples of useful mammalianhost cell lines include Chinese hamster ovary (CHO) and COS cells. Morespecific examples include monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651): human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. GenVirol., 36:59 (1977)); Chinese hamster ovary cells/-DHFR(CHO, Urlaub andChasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertali cells(TM4, Mather, Biol. Reprod., 23:243-251 (1980)); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammarytumor (MMT 060562, ATCC CCL51). The selection of the appropriate hostcell is deemed to be within the skill in the art.

[0123] In general, the choice of a mammalian host cell line for theexpression of ErbB4-Ig immunoadhesins depends mainly on the expressionvector (see below). Another consideration is the amount of protein thatis required. Milligram quantities often can be produced by transienttransfections. For example, the adenovirus EIA-transformed 293 humanembryonic kidney cell line can be transfected transiently withpRK5-based vectors by a modification of the calcium phosphate method toallow efficient immunoadhesin expression. CDM8-based vectors can be usedto transfect COS cells by the DEAE-dextran method [Aruffo et al., Cell61, 1303-1313 (1990)]; Zettmeissl et al., DNA Cell Biol. (US) 9, 347-353(1990)]. If larger amounts of protein are desired, the immunoadhesin canbe expressed after stable transfection of a host cell line. For example,a pRK5-based vector can be introduced into Chinese hamster ovary (CHO)cells in the presence of an additional plasmid encoding dihydrofolatereductase (DHFR) and conferring resistance to G418. Clones resistant toG418 can be selected in culture; these clones are grown in the presenceof increasing levels of DHFR inhibitor methotrexate; clones areselected, in which the number of gene copies encoding the DHFR andimmunoadhesin sequences is co-amplified. If the immunoadhesin contains ahydrophobic leader sequence at its N-terminus, it is likely to beprocessed and secreted by the transfected cells. The expression ofimmunoadhesins with more complex structures may require uniquely suitedhost cells; for example, components such as light chain or J chain maybe provided by certain myeloma or hybridoma cell hosts [Gascoigne etal., 1987, supra; Martin et al., J. Virol. 67, 3561-3568 (1993)].

[0124] (iii) Selection and Use of a Replicable Vector

[0125] The nucleic acid encoding immunoadhesin may be inserted into areplicable vector for cloning (amplification of the DNA) or forexpression. Various vectors are publicly available. The vector may, forexample, be in the form of a plasmid, cosmid, viral particle, or phage.The appropriate nucleic acid sequence may be inserted into the vector bya variety of procedures. In general, DNA is inserted into an appropriaterestriction endonuclease site(s) using techniques known in the art.Vector components generally include, but are not limited to, one or moreof a signal sequence, an origin of replication, one or more markergenes, an enhancer element, a promoter, and a transcription terminationsequence. Construction of suitable vectors containing one or more ofthese components employs standard ligation techniques which are known tothe skilled artisan.

[0126] The immunoadhesin may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which may be a signal sequence or other polypeptide havinga specific cleavage site at the N-terminus of the mature protein orpolypeptide. In general, the signal sequence may be a component of thevector, or it may be a part of the immunoadhesin-encoding DNA that isinserted into the vector. The signal sequence may be a prokaryoticsignal sequence selected, for example, from the group of the alkalinephosphatase, penicillinase, Ipp, or heat-stable enterotoxin II leaders.For yeast secretion the signal sequence may be, e.g., the yeastinvertase leader, alpha factor leader (including Saccharomyces andKluyveromyces α-factor leaders, the latter described in U.S. Pat. No.5,010,182), or acid phosphatase leader, the C. albicans glucoamylaseleader (EP 362,179 published Apr. 4, 1990), or the signal described inWO 90/13646 published Nov. 15, 1990. In mammalian cell expression,mammalian signal sequences may be used to direct secretion of theprotein, such as signal sequences from secreted polypeptides of the sameor related species, as well as viral secretory leaders.

[0127] Both expression and cloning vectors contain a nucleic acidsequence that enables the vector to replicate in one or more selectedhost cells. Such sequences are well known for a variety of bacteria,yeast, and viruses. The origin of replication from the plasmid pBR322 issuitable for most Gram-negative bacteria, the 2μ plasmid origin issuitable for yeast, and various viral origins (SV40, polyoma, adenovirusor BPV) are useful for cloning vectors in mammalian cells.

[0128] Expression and cloning vectors will typically contain a selectiongene, also termed a selectable marker. Typical selection genes encodeproteins that (a) confer resistance to antibiotics or other toxins,e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)complement auxotrophic deficiencies, or (c) supply critical nutrientsnot available from complex media, e.g., the gene encoding D-alanineracemase for Bacilli.

[0129] An example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theimmunoadhesin-encoding nucleic acid, such as DHFR or thymidine kinase.An appropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitableselection gene for use in yeast is the trp1 gene present in the yeastplasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1[Jones, Genetics, 85:12 (1977)].

[0130] Expression and cloning vectors usually contain a promoteroperably linked to the immunoadhesin-encoding nucleic acid sequence todirect mRNA synthesis. Promoters recognized by a variety of potentialhost cells are well known. Promoters suitable for use with prokaryotichosts include the β-lactamase and lactose promoter systems [Chang etal., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)],alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel,Nucleic Acids Res., 8:4057 (1980); EP 36,7761, and hybrid promoters suchas the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25(1983)]. Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encodingimmunoadhesin.

[0131] Examples of suitable promoter sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymees [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

[0132] Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

[0133] The transcription of immunoadhesin from vectors in mammalian hostcells is controlled, for example, by promoters obtained from the genomesof viruses such as polyoma virus, fowlpox virus (UK 2,211,504 publishedJul. 5, 1989), adenovirus (such as Adenovinus 2), bovine papillomavirus, retrovirus (such as avian sarcoma virus), cytomegalovirus,hepatitis-B virus and Simian Virus 40 (SV40); from heterologousmammalian promoters, e.g., the actin promoter or an immunoglobulinpromoter, or from heat-shock promoters, provided such promoters arecompatible with the host cell systems.

[0134] Transcription of a DNA encoding the immunoadhesin by highereukaryotes may be increased by inserting an enhancer sequence into thevector. Enhancers are cis-acting elements of DNA, usually about from 10to 300 bp, that act on a promoter to increase its transcription. Manyenhancer sequences are now known from mammalian genes (globin, elastase,albumin, α-fetoprotein, and insulin). Typically, however, one will usean enhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (by 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theimmunoadhesin coding sequence, but is preferably located at a site 5′from the promoter.

[0135] Expression vectors used in eukaryotic host cells (yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 3′ untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding immunoadhesin.

[0136] Still other methods, vectors, and host cells suitable foradaptation to the synthesis of immunoadhesin in recombinant vertebratecell culture are described in Gething et al., Nature, 293:620-625(1981); Mantei et al., Nature, 281:40-46 (1979); EP 117,060; and EP117,058.

[0137] (iv) Purification of Immunoadhesin

[0138] An immunoadhesin or a chimeric heteroadhesin preferably isrecovered from the culture medium as a secreted polypeptide, although italso may be recovered from host cell lysates. As a first step, theparticulate debris, either host cells or lysed fragments, is removed,for example, by centrifugation or ultrafiltration; optionally, theprotein may be concentrated with a commercially available proteinconcentration filter, followed by separating the chimeric heteroadhesinfrom other impurities by one or more purification procedures selectedfrom: fractionation on an immunoaffinity column; fractionation on anion-exchange column; ammonium sulphate or ethanol precipitation; reversephase HPLC; chromatography on silica; chromatography on heparinSepharose; chromatography on a cation exchange resin; chromatofocusing;SDS-PAGE; and gel filtration.

[0139] A particularly advantageous method of purifying immunoadhesins isaffinity chromatography. The choice of affinity ligand depends on thespecies and isotype of the immunoglobulin Fc domain that is used in thechimera. Protein A can be used to purify immunoadhesins that are basedon human IgG1, IgG2, or IgG4 heavy chains (Lindmark et al., J. Immunol.Meth. 62, 1-13 (1983)]. Protein G is recommended for all mouse isotypesand for human IgG3 [Guss et al., EMBO J. 5, 1567-1575 (1986)]. Thematrix to which the affinity ligand is attached is most often agarose,but other matrices are also available. Mechanically stable matrices suchas controlled pore glass or poly(styrenedivinyl)benzene allow for fasterflow rates and shorter processing times than can be achieved withagarose. The conditions for binding an immunoadhesin to the protein A orG affinity column are dictated entirely by the characteristics of the Fcdomain; that is, its species and isotype. Generally, when the properligand is chosen, efficient binding occurs directly from unconditionedculture fluid. One distinguishing feature of immunoadhesins is that, forhuman IgG1 molecules, the binding capacity for protein A is somewhatdiminished relative to an antibody of the same Fc type. Boundimmunoadhesin can be efficiently eluted either at acidic pH (at or above3.0), or in a neutral pH buffer containing a mildly chaotropic salt.This affinity chromatography step can result in an immunoadhesinpreparation that is >95% pure.

[0140] Other methods known in the art can be used in place of, or inaddition to, affinity chromatography on protein A or G to purifyimmunoadhesins. Immunoadhesins behave similarly to antibodies inthiophilic gel chromatography [Hutchens and Porath, Anal. Biochem. 159,217.226 (1986)] and immobilized metal chelate chromatography[AI-Mashikhi and Makai, J. Dairy Sci. 71, 1756-1763 (1988)]. In contrastto antibodies, however, their behavior on ion exchange columns isdictated not only by their isoelectric points, but also by a chargedipole that may exist in the molecules due to their chimeric nature.

[0141] Preparation of epitope tagged immunoadhesin, such as ErbB4-IgG,facilitates purification using an immunoaffinity column containingantibody to the epitope to adsorb the fusion polypeptide. Immunoaffinitycolumns such as a rabbit polyclonal anti-ErbB4 column can be employed toabsorb the ErbB4-IgG by binding it to an ErbB4 immune epitope.

[0142] In some embodiments, the ErbB4 receptor-immunoglobulin chimeras(immunoadhesins) are assembled as monomers, or hetero- orhomo-multimers, and particularly as dimers or tetramers, essentially asillustrated in WO 91/08298. Generally, these assembled immunoglobulinswill have known unit structures. A basic four chain structural unit isthe form in which Ig, IgD, and IgE exist. A four-unit structure isrepeated in the higher molecular weight immunoglobulins; IgM generallyexists as a pentamer of basic four units held together by disulfidebonds. IgA globulin, and occasionally IgG globulin, may also exist inmultimeric form in serum. In the case of multimer, each four unit may bethe same or different.

[0143] As noted earlier, the immunoadhesins of the present invention canbe made bispecific, and may, for example, include binding regions fromtwo different ErbB receptors, at least one or which is ErbB4. Thus, theimmunoadhesins of the present invention may have binding specificitiesfor two distinct ErbB ligands. For bispecific molecules, trimericmolecules, composed of a chimeric antibody heavy chain in one arm and achimeric antibody heavy chain-light chain pair in the other arm of theirantibody-like structure are advantageous, due to ease of purification.In contrast to antibody-producing quadromas traditionally used for theproduction of bispecific immunoadhesins, which produce a mixture of tentetramers, cells transfected with nucleic acid encoding the three chainsof a trimeric immunoadhesin structure produce a mixture of only threemolecules, and purification of the desired product from this mixture iscorrespondingly easier.

[0144] (v) Characterization of Immunoadhesin

[0145] Generally, the ErbB4 chimeric heteromultimers of the inventionwill have any one or more of the following properties: (a) the abilityto compete with a natural heteromultimeric receptor for binding to aligand such as HB-EGF; (b) the ability to form ErbB2-IgG/ErbB4-IgGcomplexes; and (c) the ability to inhibit activation of a naturalheteromultimeric receptor by depleting ligand from the environment ofthe natural receptor, thereby inhibiting proliferation of cells thatexpress the ErbB2 and ErbB4 receptor.

[0146] To screen for property (a), the ability of the chimeric ErbB4heteromultimer adhesin to bind to a ligand can be readily determined invitro. For example, immunoadhesin forms of these receptors can begenerated and the ErbB2/4-Ig heteroimmunoadhesin can be immobilized on asolid phase (e.g. on assay plates coated with goat-anti-human antibody).The ability of a ligand to bind to the immobilized immunoadhesin canthen be determined. For more details, see the ¹²⁵I-HRG binding assaydescribed in the Example below.

[0147] As to property (c), the tyrosine phosphorylation assay using MCF7cells provides a means for screening for activation of ErbB4 receptors.In an alternative embodiment of the invention, the KIRA-ELISA describedin WO 95/14930 can be used to qualitatively and quantitatively measurethe ability of an HER4 chimeric heteroadhesin to inhibit activation of aHER4 receptor.

[0148] The ability of an immunoadhesin, chimeric heteroadhesin such asErbB214-Ig, or other molecule of the present invention to inhibitproliferation of a cell that expresses the ErbB2 and ErbB4 receptor isreadily determined in cell culture by standard procedures. Useful cellsfor this experiment include MCF7 and SK-BR-3 cells obtainable from theATCC and Schwann cells (see, for example, Li et al., J. Neuroscience16(6)-0.2012-2019 (1996)). These tumor cell lines may be plated in cellculture plates and allowed to adhere thereto. The HRG ligand in thepresence and absence of a potential ErbB4 antagonist such as an ErbB4chimeric heteroadhesin is added. Monolayers are washed and stained/fixedwith crystal violet and cell growth inhibition is quantified.

[0149] 2. Antibody Preparation

[0150] Another preferred class of ErbB4 antagonists comprisesneutralizing antibodies to this receptor.

[0151] (i) Polyclonal Antibodies

[0152] Methods of preparing polyclonal antibodies are known in the art.Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. It may beuseful to conjugate the immunizing agent to a protein known to beimmunogenic in the mammal being immunized, such as serum albumin, orsoybean trypsin inhibitor. Examples of adjuvants which may be employedinclude Freund's complete adjuvant and MPL-TDM.

[0153] (ii) Monoclonal Antibodies

[0154] Monoclonal antibodies may be made using the hybridoma methodfirst described by Kohler et al., Nature, 256:495 (1975), or may be madeby recombinant DNA methods (U.S. Pat. No. 4,816,567).

[0155] In the hybridoma method, a mouse or other appropriate hostanimal, such as a hamster or macaque monkey, is immunized as hereinabovedescribed to elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the protein used forimmunization. Alternatively, lymphocytes may be immunized in vitro.Lymphocytes then are fused with myeloma cells using a suitable fusingagent, such as polyethylene glycol, to form a hybridoma cell (Goding,Monoclonal Antibodies: Principles and Practice, pp.59-103, [AcademicPress, 1986]).

[0156] The hybridoma cells thus prepared are seeded and grown in asuitable culture medium that preferably contains one or more substancesthat inhibit the growth or survival of the unfused, parental myelomacells. For example, if the parental myeloma cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (HAT medium), which substances prevent thegrowth of HGPRT-deficient cells.

[0157] Preferred myeloma cells are those that fuse efficiently, supportstable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these, preferred myeloma cell lines are murine myelomalines, such as those derived from MOP-21 and MC.-11 mouse tumorsavailable from the Salk Institute Cell Distribution Center, San Diego,Calif. USA, and SP-2 or X63-Ag8-653 cells available from the AmericanType Culture Collection, Rockville, Md. USA. Human myeloma andmouse-human heteromyeloma cell lines also have been described for theproduction of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001(1984); Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63, Marcel Dekker, Inc., New York, [1987]).

[0158] Culture medium in which hybridoma cells are growing is assayedfor production of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunosorbent assay (ELISA).

[0159] The binding affinity of the monoclonal antibody can, for example,be determined by the Scatchard analysis of Munson et al., Anal.Biochem., 107:220 (1980).

[0160] After hybridoma cells are identified that produce antibodies ofthe desired specificity, affinity, and/or activity, the cells may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, DMEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

[0161] The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

[0162] DNA encoding the monoclonal antibodies is readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of the monoclonal antibodies). The hybridomacells serve as a preferred source of such DNA. Once isolated, the DNAmay be placed into expression vectors, which are then transfected intohost cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. The DNA also may be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the homologous murine sequences,Morrison, et al., Proc. Nat. Acad. Sci. 81, 6851 (1984), or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. In thatmanner, “chimeric” or “hybrid” antibodies are prepared that have thebinding specificity of an anti-ErbB4 receptor monoclonal antibodyherein.

[0163] Typically such non-immunoglobulin polypeptides are substitutedfor the constant domains of an antibody of the invention, or they aresubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibodycomprising one antigen-combining site having specificity for a ErbB4receptor and another antigen-combining site having specificity for adifferent antigen.

[0164] Chimeric or hybrid antibodies also may be prepared in vitro usingknown methods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

[0165] Recombinant production of antibodies will be described in moredetail below.

[0166] (iii) Humanized Antibodies

[0167] Generally, a humanized antibody has one or more amino acidresidues introduced into it from a non-human source. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody.

[0168] Accordingly, such “humanized” antibodies are chimeric antibodies(Cabilly, supra), wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species. In practice, humanized antibodies are typicallyhuman antibodies in which some CDR residues and possibly some FRresidues are substituted by residues from analogous sites in rodentantibodies.

[0169] It is important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences. Threedimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e. theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequence so that thedesired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.For further details, see U.S. Pat. No. 5,821,337.

[0170] (iv) Human Antibodies

[0171] Human monoclonal antibodies can be made by the hybridoma method.Human myeloma and mouse-human heteromyeloma cell lines for theproduction of human monoclonal antibodies have been described, forexample, by Kozbor, J. Immunol. 133, 3001 (1984), and Brodeur, et al,Monoclonal Antibody Production Techniques and Applications, pp.51-63(Marcel Dekker, Inc., New York, 1987).

[0172] It is now possible to produce transgenic animals (e.g. mice) thatare capable, upon immunization, of producing a repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge. See, e.g. Jakobovits et al.,Proc. Natl. Acad. Sci. USA 90, 2551-255 (1993); Jakobovits et al.,Nature 362, 255-258 (1993).

[0173] Mendez at al. (Nature Genetics 15: 146-156 (1997]) have furtherimproved the technology and have generated a line of transgenic micedesignated as “Xenomouse II” that, when challenged with an antigen,generates high affinity fully human antibodies. This was achieved bygerm-line integration of megabase human heavy chain and light chain lociinto mice with deletion into endogenous J_(H) segment as describedabove. The Xenomouse II harbors 1,020 kb of human heavy chain locuscontaining approximately 66 V_(H) genes, complete D_(H) and J_(H)regions and three different constant regions (μ, δ and χ), and alsoharbors 800 kb of human κ locus containing 32 Vκ genes, Jκ segments andCκ genes. The antibodies produced in these mice closely resemble thatseen in humans in all respects, including gene rearrangement, assembly,and repertoire. The human antibodies are preferentially expressed overendogenous antibodies due to deletion in endogenous J_(H) segment thatprevents gene rearrangement in the murine locus.

[0174] Alternatively, the phage display technology (McCafferty et al.,Nature 348, 552-553 [1990]) can be used to produce human antibodies andantibody fragments in vitro, from immunoglobulin variable (V) domaingene repertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats; for their review see, e.g. Johnson, Kevin S. andChiswell, David J., Current Opinion in Structural Biology 3, 564-571(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., Nature 352, 624-628 (1991) isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of immunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Markset al, J. Mol. Biol. 222, 581-597 (1991), or Griffiths et al., EMBO J.12, 725-734 (1993). In a natural immune response, antibody genesaccumulate mutations at a high rate (somatic hypermutation). Some of thechanges introduced will confer higher affinity, and B cells displayinghigh-affinity surface immunoglobulin are preferentially replicated anddifferentiated during subsequent antigen challenge. This natural processcan be mimicked by employing the technique known as “chain shuffling”(Marks et al., Bio/Technol. 10, 779-783 [1992]). In this method, theaffinity of “primary” human antibodies obtained by phage display can beimproved by sequentially replacing the heavy and light chain V regiongenes with repertoires of naturally occurring variants (repertoires) ofV domain genes obtained from unimmunized donors. This techniques allowsthe production of antibodies and antibody fragments with affinities inthe nM range. A strategy for making very large phage antibodyrepertoires (also known as “the mother-of-all libraries”) has beendescribed by Waterhouse et al., Nucl. Acids Res. 21, 2265-2266 (1993),and the isolation of a high affinity human antibody directly from suchlarge phage library is reported by Griffiths et al., EMBO J. 13:3245-3260 (1994). Gene shuffling can also be used to derive humanantibodies from rodent antibodies, where the human antibody has similaraffinities and specificities to the starting rodent antibody. Accordingto this method, which is also referred to as “epitope imprinting”, theheavy or light chain V domain gene of rodent antibodies obtained byphage display technique is replaced with a repertoire of human V domaingenes, creating rodent-human chimeras. Selection on antigen results inisolation of human variable domains capable of restoring a functionalantigen-binding site, i.e. the epitope governs (imprints) the choice ofpartner. When the process is repeated in order to replace the remainingrodent V domain, a human antibody is obtained (see PCT patentapplication WO 93/06213, published Apr. 1, 1993). Unlike traditionalhumanization of rodent antibodies by CDR grafting, this techniqueprovides completely human antibodies, which have no framework or CDRresidues of rodent origin.

[0175] (i) Bispecific Antibodies

[0176] Bispecific antibodies are monoclonal, preferably human orhumanized, antibodies that have binding specificities for at least twodifferent antigens. In the present case, one of the bindingspecificities is for the ErbB4 receptor to provide an antagonistantibody, the other one is for any other antigen, and preferably foranother receptor or receptor subunit.

[0177] Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities(Millstein and Cuello, Nature 305, 537-539 (1983)). Because of therandom assortment of immunoglobulin heavy and light chains, thesehybridomas (quadromas) produce a potential mixture of 10 differentantibody molecules, of which only one has the correct bispecificstructure. The purification of the correct molecule, which is usuallydone by affinity chromatography steps, is rather cumbersome, and theproduct yields are low. Similar procedures are disclosed in PCTapplication publication No. WO93/08829 (published May 13, 1993), and inTraunecker et al., EMBO 10, 3655-3659 (1991).

[0178] According to a different and more preferred approach, antibodyvariable domains with the desired binding specificities(antibody-antigen combining sites) are fused to immunoglobulin constantdomain sequences. The fusion preferably is with an immunoglobulin heavychain constant domain, comprising at least part of the hinge, CH2 andCH3 regions. It is preferred to have the first heavy chain constantregion (CH1) containing the site necessary for light chain binding,present in at least one of the fusions. DNAs encoding the immunoglobulinheavy chain fusions and, if desired, the immunoglobulin light chain, areinserted into separate expression vectors, and are co-transfected into asuitable host organism. This provides for great flexibility in adjustingthe mutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance. In a preferred embodiment of this approach,the bispecific antibodies are composed of a hybrid immunoglobulin heavychain with a first binding specificity in one arm, and a hybridimmunoglobulin heavy chain-light chain pair (providing a second bindingspecificity) in the other arm. It was found that this asymmetricstructure facilitates the separation of the desired bispecific compoundfrom unwanted immunoglobulin chain combinations, as the presence of animmunoglobulin light chain in only one half of the bispecific moleculeprovides for a facile way of separation.

[0179] For further details of generating bispecific antibodies see, forexample, Suresh et al., Methods in Enzymology 121, 210 (1986).

[0180] (vi) Heteroconjugate Antibodies

[0181] Heteroconjugate antibodies are also within the scope of thepresent invention. Heteroconjugate antibodies are composed of twocovalently joined antibodies. Such antibodies have, for example, beenproposed to target immune system cells to unwanted cells (U.S. Pat. No.4,676,980), and for treatment of HIV infection (PCT applicationpublication Nos. WO 91/00360 and WO 92/200373; EP 03089).Heteroconjugate antibodies may be made using any convenientcross-linking methods. Suitable cross-linking agents are well known inthe art, and are disclosed in U.S. Pat. No. 4,676,980, along with anumber of cross-linking techniques.

[0182] (vii) Antibody Fragments

[0183] In certain embodiments, the ErbB4 antagonist antibody (includingmurine, human and humanized antibodies, and antibody variants) is anantibody fragment. Various techniques have been developed for theproduction of antibody fragments. Traditionally, these fragments werederived via proteolytic digestion of intact antibodies (see, e.g.,Morimoto at al., J. Biochem. Biophys. Methods 24:107-117 (1992) andBrennan et al., Science 229:81 (1985)). However, these fragments can nowbe produced directly by recombinant host cells. For example, Fab′-SHfragments can be directly recovered from E. coli and chemically coupledto form F(ab′)₂ fragments (Carter et al., Bio/Technology 10:163-167(1992)). In another embodiment, the F(ab′)₂ is formed using the leucinezipper GCN4 to promote assembly of the F(ab′)₂ molecule. According toanother approach, Fv, Fab or F(ab′)₂ fragments can be isolated directlyfrom recombinant host cell culture. Other techniques for the productionof antibody fragments will be apparent to the skilled practitioner.

[0184] (viii) Amino Acid Sequence Variants of Antibodies

[0185] Amino acid sequence variants of the ErbB4 antagonist antibodiesare prepared by introducing appropriate nucleotide changes into theErbB4 antagonist antibody DNA, or by peptide synthesis. Such variantsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of, residues within the amino acid sequences of the ErbB4antagonist antibodies of the examples shown herein. Any combination ofdeletion, insertion, and substitution is made to arrive at the finalconstruct, provided that the final construct possesses the desiredcharacteristics. The amino acid changes also may alterpost-translational processes of the humanized or variant ErbB4antagonist antibody, such as changing the number or position ofglycosylation sites.

[0186] A useful method for identification of certain residues or regionsof the ErbB4 receptor antibody that are preferred locations formutagenesis is called “alanine scanning mutagenesis,” as described byCunningham and Wells Science, 244:1081-1085 (1989). Here, a residue orgroup of target residues are identified (e.g., charged residues such asarg, asp, his, lys, and glu) and replaced by a neutral or negativelycharged amino acid (most preferably alanine or polyalanine) to affectthe interaction of the amino acids with ErbB4 receptor antigen. Thoseamino acid locations demonstrating functional sensitivity to thesubstitutions then are refined by introducing further or other variantsat, or for, the sites of substitution. Thus, while the site forintroducing an amino acid sequence variation is predetermined, thenature of the mutation per se need not be predetermined. For example, toanalyze the performance of a mutation at a given site, ala scanning orrandom mutagenesis is conducted at the target codon or region and theexpressed ErbB4 antibody variants are screened for the desired activity.

[0187] Amino acid sequence insertions include amino and/orcarboxy-terminal fusions ranging in length from one residue topolypeptides containing a hundred or more residues, as well asintrasequence insertions of single or multiple amino acid residues.Examples of terminal insertions include a ErbB4 antagonist antibody withan N-terminal methionyl residue or the antibody fused to an epitope tag.Other insertional variants of the ErbB4 antagonist antibody moleculeinclude the fusion to the N or C-terminus of the ErbB4 antagonistantibody of an enzyme or a polypeptide which increases the serum halflife of the antibody.

[0188] Another type of variant is an amino acid substitution variant.These variants have at least one amino acid residue in the ErbB4antagonist antibody molecule removed and a different residue inserted inits place. The sites of greatest interest for substitution mutagenesisinclude the hypervariable regions, but FR alterations are alsocontemplated. Conservative substitutions are shown in Table 1 under theheading of “preferred substitutions”. If such substitutions result in achange in biological activity, then more substantial changes,denominated “exemplary substitutions” in Table 1, or as furtherdescribed below in reference to amino acid classes, may be introducedand the products screened. TABLE 1 Original Exemplary Preferred ResidueSubstitutions Substitutions Ala (A) val; leu; ile val Arg (R) lys; gln;asn lys Asn (N) gln; his; asp, lys; arg gln Asp (D) glu; asn glu Cys (C)ser; ala ser Gln (Q) asn; glu asn Glu (E) asp; gln asp Gly (G) ala alaHis (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; leu phe;norleucine Leu (L) norleucine; ile; val; ile met; ala; phe Lys (K) arg;gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyrtyr Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) Tyr; phe tyrTyr (Y) Trp; phe; thr; ser phe Val (V) Ile; leu; met; phe; leu ala;norleucine

[0189] Substantial modifications in the biological properties of theantibody are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

[0190] (1) hydrophobic: norleucine, met, ala, val, leu, ile;

[0191] (2) neutral hydrophilic: cys, ser, thr;

[0192] (3) acidic: asp, glu;

[0193] (4) basic: asn, gln, his, lys, arg;

[0194] (5) residues that influence chain orientation: gly, pro; and

[0195] (6) aromatic: trp, tyr, phe.

[0196] Non-conservative substitutions will entail exchanging a member ofone of these classes for another class.

[0197] Any cysteine residue not involved in maintaining the properconformation of the ErbB4 antagonist antibody also may be substituted,generally with serine, to improve the oxidative stability of themolecule and prevent aberrant crosslinking. Conversely, cysteine bond(s)may be added to the antibody to improve its stability (particularlywhere the antibody is an antibody fragment such as a Fv fragment).

[0198] A particularly preferred type of substitution variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g. a humanized or human antibody). Generally, the resultingvariant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionvariants is affinity maturation using phage display. Briefly, severalhypervariable region sites (e.g. 6-7 sites) are mutated to generate allpossible amino substitutions at each site. The antibody variants thusgenerated are displayed in a monovalent fashion from filamentous phageparticles as fusions to the gene III product of M13 packaged within eachparticle. The phage-displayed variants are then screened for theirbiological activity (e.g. antagonist activity) as herein disclosed. Inorder to identify candidate hypervariable region sites for modification,alanine scanning mutagenesis can be performed to identify hypervariableregion residues contributing significantly to antigen binding.Alternatively, or in addition, it may be beneficial to analyze a crystalstructure of the antigen-antibody complex to identify contact pointsbetween the antibody and ErbB receptor. Such contact residues andneighboring residues are candidates for substitution according to thetechniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

[0199] Another type of amino acid variant of the antibody alters theoriginal glycosylation pattern of the antibody. By altering is meantdeleting one or more carbohydrate moieties found in the antibody, and/oradding one or more glycosylation sites that are not present in theantibody.

[0200] Glycosylation of antibodies is typically either N-linked orO-linked. N-linked refers to the attachment of the carbohydrate moietyto the side chain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except praline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

[0201] Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

[0202] Nucleic acid molecules encoding amino acid sequence variants ofthe ErbB4 antagonist antibodies are prepared by a variety of methodsknown in the art. These methods include, but are not limited to,isolation from a natural source (in the case of naturally occurringamino acid sequence variants) or preparation by oligonucleotide-mediated(or site-directed) mutagenesis, PCR mutagenesis, and cassettemutagenesis of an earlier prepared variant or a non-variant version ofthe ErbB4 antagonist antibody.

[0203] (ix) Other Modifications of Antibodies

[0204] The ErbB4 antagonist antibodies disclosed herein may also beformulated as immunoliposomes. Liposomes containing the antibody areprepared by methods known in the art, such as described in Epstein etal., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc.Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and4,544,545. Liposomes with enhanced circulation time are disclosed inU.S. Pat. No. 5,013,556.

[0205] Particularly useful liposomes can be generated by the reversephase evaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem. 257:286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al, J. National Cancer Inst.81(19):1484 (1989).

[0206] The antibody of the present invention may also be used in ADEPTby conjugating the antibody to a prodrug-activating enzyme whichconverts a prodrug (e.g., a peptidyl chemotherapeutic agent, seeWO81/01145) to an active anti-cancer drug. See, for example, WO 88/07378and U.S. Pat. No. 4,975,278.

[0207] The enzyme component of the immunoconjugate useful for ADEPTincludes any enzyme capable of acting on a prodrug in such a way so asto covert it into its more active, cytotoxic form.

[0208] Enzymes that are useful in the method of this invention include,but are not limited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents; carbohydratecleaving enzymes such as -galactosidase and neuraminidase useful forconverting glycosylated prodrugs into free drugs; -lactamase useful forconverting drugs derivatized with -lactams into free drugs; andpenicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs. Alternatively, antibodies with enzymatic activity, alsoknown in the art as “abzymes”, can be used to convert the prodrugs ofthe invention into free active drugs (see, e.g., Massey, Nature328:457-458 (1987)). Antibody-abzyme conjugates can be prepared asdescribed herein for delivery of the abzyme to a tumor cell population.

[0209] The enzymes of this invention can be covalently bound to theErbB4 antagonist antibodies by techniques well known in the art such asthe use of the heterobifunctional crosslinking reagents discussed above.Alternatively, fusion proteins comprising at least the antigen bindingregion of an antibody of the invention linked to at least a functionallyactive portion of an enzyme of the invention can be constructed usingrecombinant DNA techniques well known in the art (see, e.g., Neubergeret al., Nature 312:604-608 [1984]).

[0210] In certain embodiments of the invention, it may be desirable touse an antibody fragment, rather than an intact antibody. In this case,it may be desirable to modify the antibody fragment in order to increaseits serum half-life. This may be achieved, for example, by incorporationof a salvage receptor binding epitope into the antibody fragment (e.g.,by mutation of the appropriate region in the antibody fragment or byincorporating the epitope into a peptide tag that is then fused to theantibody fragment at either end or in the middle, e.g., by DNA orpeptide synthesis). See WO96/32478 published Oct. 17, 1996.

[0211] The salvage receptor binding epitope generally constitutes aregion wherein any one or more amino acid residues from one or two loopsof a Fc domain are transferred to an analogous position of the antibodyfragment. Even more preferably, three or more residues from one or twoloops of the Fc domain are transferred. Still more preferred, theepitope is taken from the CH2 domain of the Fc region (e.g., of an IgG)and transferred to the CH1, CH3, or V_(H) region, or more than one suchregion, of the antibody. Alternatively, the epitope is taken from theCH2 domain of the Fc region and transferred to the C_(L) region or V_(L)region, or both, of the antibody fragment.

[0212] Covalent modifications of the ErbB4 antagonist antibodies arealso included within the scope of this invention. They may be made bychemical synthesis or by enzymatic or chemical cleavage of the antibody,if applicable. Other types of covalent modifications of the antibody areintroduced into the molecule by reacting targeted amino acid residues ofthe antibody with an organic derivatizing agent that is capable ofreacting with selected side chains or the N- or C-terminal residues.Exemplary covalent modifications of polypeptides are described in U.S.Pat. No. 5,534,615, specifically incorporated herein by reference. Apreferred type of covalent modification of the antibody compriseslinking the antibody to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, inthe manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

[0213] 3. Preparation of Soluble ErbB4 Receptors

[0214] Soluble ErbB4 receptors, such an ErbB4 extracellular domain, canbe prepared by culturing cells transformed or transfected with a vectorcontaining the encoding nucleic acid. It is, of course, contemplatedthat alternative methods, which are well known in the art, may beemployed to prepare such soluble receptors. For instance, the solublereceptor sequence, or portions thereof, may be produced by directpeptide synthesis using solid-phase techniques (see, e.g., Stewart etal., supra; and Merrifield, supra). In vitro protein synthesis may beperformed using manual techniques or by automation. Automated synthesismay be accomplished, for instance, using an Applied Biosystems PeptideSynthesizer (Foster City, Calif.) using manufacturer's instructions.Various portions of the soluble receptor may be chemically synthesizedseparately and combined using chemical or enzymatic methods to producethe full-length soluble receptor.

[0215] Recombinant production of soluble ErbB4 receptors is performedessentially as described hereinabove in connection with immunoadhesins.

[0216] The most convenient method for the large-scale production ofsoluble ErbB4 receptors is by cleavage from an ErbB4-Ig immunoadhesin.The structural similarity between immunoadhesins and antibodiessuggested that it might be possible to cleave immunoadhesins byproteolytic enzymes such as papain, to generate Fd-like fragmentscontaining the “adhesin” portion. In order to provide a more genericapproach for cleavage of immunoadhesins, proteases which are highlyspecific for their target sequence are to be used. A protease suitablefor this purpose is an engineered mutant of subtilisin BPN, whichrecognizes and cleaves the sequence AAHYTL. Introduction of this targetsequence into the support hinge region of an ErbB4-IgG (e.g. IgG1)immunoadhesin facilitates highly specific cleavage between the Fc andtrk domains. The IgG1 immunoadhesin is purified by protein Achromatography and cleaved with an immobilized form of the enzyme.Cleavage results in two products; the Fc region and the ErbB4 region,which is preferably an ErbB4 extracellular domain. These fragments canbe separated easily by a second passage over a protein A column toretain the Fc and obtain the purified ErbB4 fragment in the flow-throughfractions. A similar approach can be used to generate a dimeric ErbB4portion, by placing the cleavable sequence at the lower hinge.

[0217] 4. Therapeutic Compositions and Methods

[0218] The members of the ErbB family of receptors and correspondingligands are involved in smooth muscle cell proliferation in variousorgans. Accordingly, an ErbB4 receptor antagonist may be utilized forthe treatment of a variety of “diseases or disorders” involving smoothmuscle cell proliferation in a mammal, such as a human.

[0219] In a preferred embodiment, the present invention concerns the useof ErbB4 receptor antagonists for the treatment of cardiac diseasesinvolving proliferation of vascular smooth muscle cells (VSMC) andleading to intimal hyperplasia such as vascular stenosis, restenosisresulting from angioplasy or surgery or stent implants, atherosclerosisand hypertension (reviewed in Casterella and Teirstein, Cardiol. Rev. 7:219-231 [1999]; Andres, Int. J. Mol. Med. 2: 81-89 [1998]; and Rosanioet al., Thromb Haemost. 82 [suppl 1]: 164-170 [1999]). There is anintricate interplay of various cells and cytokines released that act inautocrine, paracrine or juxtacrine manner, which result in migration ofVSMCs from their normal location in media to the damaged intima. Themigrated VSMCs proliferate excessively and lead to thickening of intima,which results in stenosis or occlusion of blood vessels. The problem iscompounded by platelet aggregation and deposition at the site of lesion.α-thrombin, a multifunctional serine protease, is concentrated at siteof vascular injury and stimulates VSMCs proliferation. Followingactivation of this receptor, VSMCs produce and secrete various autocrinegrowth factors, including PDGF-AA, HB-EGF and TGF-(3 (reviewed inStouffer and Runge, Semin. Thromb. Hemost. 24: 145-150 [1998]).

[0220] Various members of the EGF family play important roles in thenormal growth and maintenance of blood vessels as well as inpathological conditions. For example, heparin-binding EGF-like growthfactor (HB-EGF) is a potent mitogen and a chemotactic factor forfibroblasts as well as VSMCs but not endothelial cells (reviewed in Raaband Klagsbrun, Biochim. Biophys. Acta 1333: F179-199 [1997]). Vascularendothelial growth factor (VEGF), a powerful angiogenic factor, inducesthe expression of HB-EGF in vascular endothelial cells (Arkonac et al.,J. Biol. Chem. 273: 4400-4405 [1998]). HB-EGF binds to and activatesHER1 and ErbB4 receptors initiating a signal transduction cascade thatultimately results in migration and proliferation of fibroblasts andVSMCs. HB-EGF also stimulates VSMCs to secrete various factors that aremitogenic for endothelial cells (Abramovitch at al., FEBS Lett. 425:441-447 [1998]). Moreover, it also induces chemotactic response inendothelial cells. Similarly, another ligand that activates EGFreceptors, epiregulin, is secreted by VSMCs stimulated with angiotensinII, endothelin-1 and thrombin, and also acts as a powerful mitogen forproliferation of VSMCs (Taylor et al., Proc. Natl. Acad. Sci. USA 96:1633-1638 [1999]).

[0221] Vascular stenosis gives rise to hypertension as a result ofincreased resistance to blood flow. Moreover, decreased blood supply tothe tissue may also cause necrosis and induce inflammatory responseleading to severe damage. For example, myocardial infarction occurs as aresult of lack of oxygen and local death of heart muscle tissues.Percutaneous transluminal coronary angioplasy (PTCA), simply referred toas balloon angioplasty, is a non-surgical catheter-based treatment forobstructive coronary artery disease. In this method, a catheter isintroduced in the blood vessel and a balloon is inflated at the site ofplaque in order to mechanically dislodge the plaque. Alternatively,stent is implanted to restore smooth blood flow. However, neointimalformation takes place even within the implanted stent, known as“in-stent restenosis.” For example, stent deployment results in earlythrombus deposition and acute inflammation, granulation tissuedevelopment, and ultimately smooth muscle cell proliferation andextracellular matrix synthesis (reviewed in Virmani and Farb, Curr.Opin. Lipidol. 10: 499-506 [1999]). Bypass surgery is performed to getaround the affected blood vessel only in severe cases, and usually onlyafter multiple rounds of angioplasty have failed in restoring bloodflow.

[0222] Although balloon angioplasty has been used widely for thetreatment of stenosis, its long-term success is limited by restenosis.Restenosis persists as the limiting factor in the maintenance of vesselpatency after PTCA, occurring in 30-50% of patients and accounting forsignificant morbidity and health care expenditure. The underlyingmechanisms of restenosis are comprised of a combination of effects fromvessel recoil, negative vascular remodeling, thrombus formation andneointimal hyperplasia. Importantly, these events are interconnected.For example, neointimal hyperplasia is stimulated by growth factors,which are released by local thrombi and the injured arterial segmentitself, and act to enhance the expression of other growth-stimulatingproteins resulting in acute proliferative and inflammatory responses.For instance, endothelial injury induces expression of EGF, EGF-likefactors and EGFR in VSMCs, which act upon them in an autocrine manner tostimulate their proliferation leading to intimal thickening andrestenosis. Extracellular matrix (ECM) formation and accumulation in thevessel wall is another important component of the restenosis lesion thatdevelops after balloon angioplasty.

[0223] A multitude of pharmacological trials have been conducted in anattempt to prevent restenosis, but most have demonstrated littlebenefits. Early clinical trials in restenosis prevention using variousrevascularization devices, anti-platelet drugs, anti-thrombotic drugsand anti-inflammatory drugs were uniformly negative (reviewed inCasterella and Teirstein, Cardiol. Rev. 7: 219-231 [1999]; Andres, Int.J. Mol. Med. 2: 81-89 [1998]; and Rosanio et al., Thromb. Haemost. 82[suppl 1]: 164-170 [1999]). Inspite of all the recent progress, there isstill no satisfactory treatment for stenosis or prevention of restenosisafter balloon angioplasty or stent implantation. Although limitedsuccess has been achieved in small randomized trials, stenosis, andparticularly restenosis, remains a major clinical problem. The instantinvention discloses the use of ErbB4 receptor antagonists for thetreatment of stenosis or restenosis by controlling the proliferation ofvascular smooth muscle cells.

[0224] The scope of the present invention, however, is not restricted tothe disorders of the vascular smooth muscle cells. The scopespecifically includes any disorder that results from proliferation ofsmooth muscle cells in any organ and that involves an active role ofErbB4 receptors and/or corresponding ligands.

[0225] Infantile hypertrophic pyloric stenosis (IHPS) is a relativelycommon disease that primarily affects young infants. The underlyingstenosis causes functional obstruction of the pyloric canal.Consequently, gastric emptying of milk is disturbed severely. IHPSinvolves hypertrophy and hyperplasia of the pyloric smooth muscle massand results in pyloric stenosis (Oue and Puri, Pediatr. Res. 45: 853-857[1999]). Furthermore, increased expression of EGF, EGF receptor andHB-EGF has been reported, in SMCs in pyloric circular and longitudinalmuscle from IHPS patients as compared to control tissues (Shima et al.,Pediatr. Res. 47: 201-207 [2000]). The antagonists of ErbB4 disclosedherein may find use in the control of pyloric smooth muscle cellproliferation and therefore in the treatment of pyloric stenosis.

[0226] The contractile nature of smooth muscle cells and regulation oftheir contraction by various factors play a crucial role in the urinarycollecting system including bladder, ureters and urethra. Amembrane-bound precursor form of HB-EGF is expressed in urinary bladdersmooth muscle cells and epithelial cells (Freeman et al., J. Clin.Invest. 99: 1028-1036 [1997]; Kaefer et al., J. Urol. 163: 580-584[2000]). Moreover, treatment of bladder SMCs with diphtheria toxin,which is known to utilize membrane-bound HB-EGF as a receptor, inhibitedtheir proliferation (Kaefer et al., ibid). HB-EGF is a potent mitogenfor bladder SMC proliferation, and it acts by binding to ErbB1 (HER1)receptors expressed by these cells, thus acting as an autocrine growthfactor (Borer et al., Lab Invest. 79: 1335-1345 [1999]). The authorsalso demonstrated the expression of ErbB2 and ErbB3 but not ErbB4receptors on bladder SMCs. These findings raise the possibility thatHB-EGF plays a role in the bladder wall thickening that occurs inresponse to obstructive syndromes affecting the lower urinary tract.Therefore, ErbB4 antagonists of the instant invention, particularlyErbB4 immunoadhesin, may prove useful in controlling proliferation ofbladder smooth muscle cells, and consequently in the prevention ortreatment of urinary obstructive syndromes.

[0227] The obstructive airway diseases are yet another group of diseaseswith underlying pathology involving smooth muscle cell proliferation.One example of this group is asthma which manifests in airwayinflammation and bronchoconstriction. EGF has been shown to stimulateproliferation of human airway SMCs and is likely to be one of thefactors involved in the pathological proliferation of airway SMCs inobstructive airway diseases (Cerutis et al., Am. J. Physiol. 273: L10-15[1997]; Cohen et al., Am. J. Respir. Cell. Mol. Biol. 16: 85-90 [1997]).Accordingly, the ErbB4 antagonists of the present invention may be usedfor the treatment of obstructive airway diseases.

[0228] There are two major approaches to introducing the nucleic acid(optionally contained in a vector) into the patient's cells; in vivo andex vivo. For in vivo delivery the nucleic acid is injected directly intothe patient, usually at the site where the chimeric heteroadhesin isrequired. For ex vivo treatment, the patient's cells are removed, thenucleic acid is introduced into these isolated cells and the modifiedcells are administered to the patient either directly or, for example,encapsulated within porous membranes which are implanted into thepatient (see, e.g. U.S. Pat. Nos. 4,892,538 and 5,283,187).

[0229] There are a variety of techniques available for introducingnucleic acids into viable cells. The techniques vary depending uponwhether the nucleic acid is transferred into cultured cells in vitro, orin vivo in the cells of the intended host. Techniques suitable for thetransfer of nucleic acid into mammalian cells in vitro include the useof liposomes, electroporation, microinjection, cell fusion,DEAE-dextran, the calcium phosphate precipitation method, etc.

[0230] A commonly used vector for ex vivo delivery of the gene is aretrovirus. The currently preferred in vivo nucleic acid transfertechniques include transfection with viral vectors (such as adenovirus,Herpes simplex I virus, or adeno-associated virus) and lipid-basedsystems (useful lipids for lipid-mediated transfer of the gene areDOTMA, DOPE and DC-Chol, for example). In some situations it isdesirable to provide the nucleic acid source with an agent that targetsthe target cells, such as an antibody specific for a cell surfacemembrane protein or the target cell, a ligand for a receptor on thetarget cell, etc. Where liposomes are employed, proteins which bind to acell surface membrane protein associated with endocytosis may be usedfor targeting and/or to facilitate uptake, e.g. capsid proteins orfragments thereof tropic for a particular cell type, antibodies forproteins which undergo internalization in cycling, and proteins thattarget intracellular localization and enhance intracellular half-life.The technique of receptor-mediated endocytosis is described, forexample, by Wu et al, J. Biol. Chem. 262:4429-4432 (1987); and Wagner etal, Proc. Natl. Acad. Sci. USA 87:3410-3414 (1990). For review of thecurrently known gene marking and gene therapy protocols see Anderson etal., Science 256:808-813 (1992). See also WO 93125673 and the referencescited therein.

[0231] Therapeutic formulations are prepared for storage by mixing theErbB4 antagonist having the desired degree of purity with optionalphysiologically acceptable carriers, excipients, or stabilizers(Remington's Pharmaceutical Sciences, 16th Edition, Osol., A., Ed.,(1980)), in the form of lyophilized cake or aqueous solutions.Pharmaceutically acceptable carriers, excipients, or stabilizers arenon-toxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween, Pluronics, or polyethylene glycol (PEG).

[0232] An antibody or an immunoadhesin to be used for in vivoadministration must be sterile. This is readily accomplished byfiltration through sterile filtration membranes, prior to or followinglyophilization and reconstitution. The formulation ordinarily will bestored in lyophilized form or in solution.

[0233] Therapeutic compositions are generally placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

[0234] The route of antibody, immunoadhesin or chimeric heteroadhesinadministration is in accord with known methods, e.g., injection orinfusion by intravenous, intraperitoneal, intracerebral, intramuscular,intraocular, intraarterial, or intralesional routes, or bysustained-release systems as noted below. The heteroadhesin or antibodyis administered continuously by infusion or by bolus injection.

[0235] Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theprotein, which matrices are in the form of shaped articles, e.g., films,or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., J. Biomed. Mater. Res., 15:167-277 (1981)and Langer, Chem. Tech., 12:98-105 (1982) or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers,22:547-556 (1983)), non-degradable ethylene-vinyl acetate (Langer etal., supra), degradable lactic acid-glycolic acid copolymers such as theLupron Depot (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid (EP 133,988).

[0236] Sustained-release ErbB4 antagonist also include liposomallyentrapped drug. Liposomes containing ErbB4 antagonist are prepared bymethods known per se: Epstein at al., Proc. Natl. Acad. Sci. USA82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP142,641; Japanese patent application 83-118008; U.S. Pat. Nos. 4,485,045and 4,544,545; and EP 102,324. Ordinarily the liposomes are of the small(about 200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol. % cholesterol, the selected proportion beingadjusted for the optimal therapy. Particularly useful liposomes can begenerated by the reverse phase evaporation method with a lipidcomposition comprising phosphatidylcholine, cholesterol andPEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes areextruded through filters of defined pore size to yield liposomes withthe desired diameter. A chemotherapeutic agent (such as Doxorubicin) isoptionally contained within the liposome. See Gabizon et al. J. NationalCancer Inst. 81(19):1484 (1989).

[0237] The ErbB4 antagonist of the invention may be used to bind andsequester ErbB4 ligand or block ErbB4 receptor thereby inhibiting ErbB4activation in the cell and inhibit cell proliferation. The ErbB4antagonist of the invention may be administered to a patient along withother therapy such as a chemotherapeutic agent. Preparation and dosingschedules for such chemotherapeutic agents may be used according tomanufacturers' instructions or as determined empirically by the skilledpractitioner. Preparation and dosing schedules for such chemotherapy arealso described in Chemotherapy Service Ed., M. C. Perry, Williams &Wilkins, Baltimore, Md. (1992). The chemotherapeutic agent may precede,or follow administration of the antagonist or may be givensimultaneously therewith.

[0238] An effective amount of antagonist to be employed therapeuticallywill depend, for example, upon the therapeutic objectives, the route ofadministration, and the condition of the patient. Accordingly, it willbe necessary for the therapist to titer the dosage and modify the routeof administration as required to obtain the maximum therapeutic effect.A typical dosage might range from about 1 g/kg to up to 100 mg/kg ofpatient body weight, preferably about 10 g/kg to 10 mg/kg. Typically,the clinician will administer antagonist until a dosage is reached thatachieves the desired effect for treatment of the above mentioneddisorders.

[0239] 5. Methods for Identification of Molecules that Inhibit orEnhance the Proliferation or Migration of Smooth Muscle Cells

[0240] The present invention discloses a method of screening to identifymolecules that can inhibit or enhance the proliferation of smooth musclecells. For example, a candidate molecule is incubated with a polypeptidecomprising the extracellular domain of an ErbB4 receptor, followed byadding to a culture of smooth muscle cells and determining the effect onthe proliferation of cells. The ErbB4 receptor may be a native ErbB4receptor such as a human ErbB4 receptor, or may be a polypeptide havingat least 85% sequence identity with the amino acid sequence of a nativeErbB4 receptor. The cell proliferation can be monitored and quantitatedin a number of ways. For instance, incorporation of ³H-thymidine intoDNA is a well-established method to monitor cellular DNA synthesisindicative of cell proliferation. The incorporation of ³H-thymidine intoDNA is monitored either microscopically by counting the number of silvergrains in an autoradiograph or biochemically by liquid scintillationcounting. Similarly, incorporation of 5-bromo 2′-deoxyuridine (BrdU)into cellular DNA can be monitored either microscopically orimmunologically. Both assays utilize highly specific monoclonalantibodies that recognize BrdU incorporated into DNA. In the microscopicassay, the cells are permeabilized, reacted with BrdU specificmonoclonal antibodies followed by labeled secondary antibodies. Thesecondary antibodies are detected by virtue of an attached label such asa fluorescent dye (fluorescein isothiocyanate (FITC), rhodamine, TexasRed etc) or an enzymatic label (alkaline phosphatase, horseradishperoxidase etc). A suitable substrate that produces an insoluble productupon enzymatic action is then used to reveal and quantitate the enzymelabeled secondary antibodies. An enzymatic assay monitors the amount ofBrdU specific monoclonal antibodies by a suitable immunoassay such asELISA. The monoclonal antibodies specific for BrdU as well as ELISA kitscontaining such antibodies are available commercially from a number ofsources including Boehringer Mannheim. A flow cytometry can also be usedto monitor cell proliferation. In this method, cells are fractionatedbased on the nuclear DNA content per cell. Since the nuclear DNA contentvaries among cells undergoing division depending on the phase of cellcycle (2n in G1 phase, 4n in G2+M phase and intermediate value in Sphase, wherein n is the value of haploid nuclear DNA content), cellproliferation can be rapidly monitored by estimating the fraction ofcells in S and G2+M phases using this approach.

[0241] Since ErbB4-dependent proliferation of smooth, muscle cellsinvolves ligand-mediated signal transduction pathway utilizing ErbB4receptor, any step in this pathway can be monitored and used as ameasure of cell proliferation. One such step is a ligand-inducedtyrosine autophosphorylation of ErbB4 receptor, which can be monitoredby the kinase receptor activation (KIRA) assay as described inWO95/14930. This ELISA-type assay is suitable for qualitative orquantitative measurement of kinase activation by measuring theautophosphorylation of the kinase domain of a receptor protein tyrosinekinase such as ErbB4. The first stage of the assay involvesphosphorylation of the kinase domain of ErbB4 receptor present in thecell membrane of a smooth muscle cell. Typically, a first solid phase(e.g., a well of a first assay plate) is coated with a substantiallyhomogeneous population of smooth muscle cells. Being adherent cells, thesmooth muscle cells adhere naturally to the first solid phase. One canalso use smooth muscle cells transfected with a “receptor construct”that comprises a fusion of a kinase receptor and a flag polypeptide.Antibodies specific for flag polypeptide are used in the ELISA part ofthe assay to capture the receptor with flag peptide. A candidatemolecule and a polypeptide comprising the extracellular domain of anative ErbB4 receptor are then added to the wells containing smoothmuscle cells, followed by monitoring tyrosine autophosphorylation ofErbB4 receptor by the KIRA assay. A polypeptide comprising an amino acidsequence having at least 85% sequence identity with the amino acidsequence of the extracellular domain of ErbB4 receptor can also be usedin the assay. Following exposure, the smooth muscle cells aresolubilized using a lysis buffer (which has a solubilizing detergenttherein) and gentle agitation, thereby releasing cell lysate which canbe subjected to the ELISA part of the assay directly, without the needfor concentration or clarification of the cell lysate.

[0242] The cell lysate thus prepared is then subjected to the second(ELISA) stage of the assay. As a first step in the ELISA stage, a secondsolid phase (usually a well of an ELISA microtiter plate) is coated witha capture agent (often a capture antibody) which binds specifically toErbB4 receptor or, in the case of a receptor construct, to the flagpolypeptide. Coating of the second solid phase is carried out so thatthe capture agent adheres to the second solid phase. The capture agentis generally a monoclonal antibody but polyclonal antibodies may also beused. The cell lysate obtained is then exposed to, or contacted with,the adhering capture agent so that the receptor or receptor constructadheres to (or is captured in) the second solid phase. A washing step isthen carried out, so as to remove unbound cell lysate, leaving thecaptured receptor or receptor construct. The adhering or capturedreceptor or receptor construct is then exposed to, or contacted with, ananti-phosphotyrosine antibody which identifies phosphorylated tyrosineresidues in the tyrosine kinase receptor. In the preferred embodiment,the anti-phosphotyrosine antibody is conjugated (directly or indirectly)to an enzyme which catalyses a color change of a non-radioactive colorreagent. Accordingly, phosphorylation of the receptor can be measured bya subsequent color change of the reagent. The enzyme can be bound to theanti-phosphotyrosine antibody directly, or a conjugating molecule (e.g.,biotin) can be conjugated to the anti-phosphotyrosine antibody and theenzyme can be subsequently bound to the anti-phosphotyrosine antibodyvia the conjugating molecule. Finally, binding of theanti-phosphotyrosine antibody to the captured receptor or receptorconstruct is measured, e.g., by a color change in the color reagent.Anti-phosphotyrosine antibodies that are commercially available can beused for the assay.

[0243] The instant invention also provides for a method for screening ofmolecules which can inhibit or enhance migration of smooth muscle cells.One of the formats utilizes a compartmentalized chemotaxis cell culturechambers such as Neuroprobe ChemoTX chemotaxis chambers available from(Neuroprobe Inc., Gaithersburg, Md.). In this method, a porous filterseparates smooth muscle cells in the upper chamber from a mediumcontaining a chemoattractant (e.g. thrombin) in the lower chamber.Smooth muscle cells are incubated with a candidate molecule and apolypeptide comprising the extracellular domain of an ErbB4 receptor. Atthe end of incubation period, the filters are stained and smooth musclecells that have migrated to the bottom of the filter are counted usingan inverted microscope.

[0244] A conventional library or a combinatorial library of chemicalcompounds can be used for screening purpose. An automated approachadapted for high throughput can be conveniently used for the assay.However, the screening assays are not restricted only to smallmolecules, even macromolecules such as antibodies can be used for thescreening.

EXAMPLES

[0245] The following examples are offered by way of illustration and notby way of limitation. The examples are provided so as to provide thoseof ordinary skill in the art with a complete disclosure and descriptionof how to make and use the compounds, compositions, and methods of theinvention and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to insure accuracywith respect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviation should be accounted for. Unlessindicated otherwise, parts are parts by weight, temperature is indegrees C., and pressure is at or near atmospheric. The disclosures ofall citations in the specification are expressly incorporated herein byreference.

Example 1 Construction, Isolation and Biochemical Characterization ofImmunoadhesins and Chimeric Heteromultimer Immunoadhesins

[0246] A unique Mlu I site was engineered into a plasmid expressinghuman IgG heavy chain (pDR, a gift from J. Ridgeway and P. Carter,Genentech, Inc.) at the region encoding the hinge domain of theimmunoglobulin. Mlu I sites were also engineered into a set of ErbB4expression plasmids at the region encoding the ECD/TM junctions of thesereceptors. All mutagenesis were performed using the Kunkel method(Kunkel, T., Proc. Natl. Acad. Sci. U.S.A. 82:488 (1985)). The Mlu Isites were utilized to make the appropriate ErbB4-IgG fusion constructs.The fusion junction of the ErbB-IgG chimera was: G⁶⁴⁰_(ErbB4)-(TR)-DKTH²²⁴ _(VH), where the amino acid numbering of the ErbB4polypeptide is described in Plowman et al. (Plowman, G. D. et al.,(1993a) PNAS USA 90:1746-1750) The conserved TR sequence is derived fromthe Mlu I site. The sequence of the Fc region used in the preparation ofthe fusion constructs is found in Ellison, J. W. et al (Ellison, J. W.et al. (1982) NAR 10:4071-4079). The final expression constructs were ina pRK-type plasmid backbone wherein eukaryotic expression is driven by aCMV promoter (Gorman et al., DNA Prot. Eng. Tech. 2:3-10 (1990)).

[0247] To obtain protein for in vitro experiments, adherent HEK-293cells (ATCC No. CRL-1573) were transfected with the expression plasmidusing standard calcium phosphate methods (Gorman et al., supra and Huangat al., Nucleic Acids Res. 18:937-947 (1990)). Serum-containing mediawas replaced with serum-free media 15 hours post-transfection and thetransfected cells incubated for 5-7 days. The resulting conditionedmedia was harvested and passed through Protein A columns (1 ml PharmaciaHiTrap). Purified IgG fusions were eluted with 0.1 M citric acid (pH4.2) into tubes containing 1 M Tris pH 9.0. The eluted proteins weresubsequently dialyzed against PBS and concentrated using Centri-prep-30filters (Amicon). Glycerol was added to a final concentration of 25% andthe material stored at −20 C. Concentrations of material were determinedvia a Fc-ELISA

[0248]¹²⁵I-HRG Binding Assay

[0249] The EGF-like domain of HRG 1₍₁₇₇₋₂₄₄₎ was expressed in E. coli,purified and radioiodinated as described previously (Sliwkowski, M. etal. J. Biol. Chem. 269:14661-14665 (1994)). Full-length rHRG 1, whichwas expressed in Chinese hamster ovary cells, was used in Western blotanalysis. Binding assays were performed in Nunc breakapart immuno-moduleplates. Plate wells were coated at 4 C overnight with 100 l of 5 g/mlgoat-anti-human antibody (Boehringer Mannheim) in 50 mM carbonate buffer(pH 9.6). Plates were rinsed twice with 200 l wash buffer (PBS/0.05%Tween-20) followed by a brief incubation with 100 l 1% BSA/PBS for 30min at room temperature. Buffer was removed and each well was incubatedwith 100 l IgG fusion protein in 1% BSA/PBS under vigorous side-to-siderotation for 1 hour. Plates were rinsed three times with wash buffer andcompetitive binding was carried out by adding various amounts of coldcompetitor -HRG and ¹²⁵I-HRG 1 and incubating at room temperature for2-3 hours with vigorous side-to-side rotation. Wells were quickly rinsedthree times with wash buffer, drained and individual wells were countedusing a 100 Series Iso Data-counter. Scatchard analysis was performedusing a modified Ligand program (Munson, P. and Robard, D. (1980)Analytical Biochemistry 107:220-239).

[0250]³H-Thymidine Incorporation Assay

[0251] Tritiated thymidine incorporation assays were performed in a96-well format. MCF7 cells were plated at 10,000 cells/well in 50:50F12/DMEM (high glucose) 0.1% fetal calf serum (100 ml). Cells wereallowed to settle for 3 hours, after which ErbB4-IgG fusion proteinsand/or heregulin were added to the wells (final volume of 200 ml) andthe plates incubated for 15 hours in a 37° C. tissue culture incubator.Tritiated thymidine was added to the wells (20 ml of 1120 dilutedtritiated thymidine stock: Amersham TRA 120 B363, 1 mCi/ml) and theplates incubated a further 3 hours. Tritiated material was thenharvested onto GF/C unifilters (96 well format) using a PackardFiltermate 196 harvester. Filters were counted using a Packard Topcountapparatus.

Example 2 Effect of ErbB4-IgG Immunoadhesin on Human Aortic SmoothMuscle Cell Proliferation

[0252] Human aortic smooth muscle cells (Clonetics) were seeded at about50% confluent density (5000 cells/well) in 96 well tissue culture platesand incubated overnight in SM2 media (Clonetics). Next day, the mediawas changed to M199 supplemented with ITS (1×), 2 mM L-glutamine, 50μg/ml ascorbic acid, 26.5 mM NaHCO3, 100 U/ml penicillin, 100 U/mlstreptomycin and 0.1% (v/v) fetal bovine serum. The cells were furtherincubated for 16 h. The cells were then treated with either Her4-IgG(400 nM) or buffer for 1 h, followed by treatment with PDGF (100 ng/ml)for 40 h. Control cells were left untreated to estimate the basal levelof cell growth. An aliquot of BrdU (10 μl/well of a 10 μM solution of5-bromo 2′-deoxyuridine prepared in PBS) was added and the cells wereincubated for an additional 2 h. Cell proliferation was monitored byquantitating BrdU incorporation using BrdU ELISA (Cell proliferationkit, Boehringer mannheim, Catalog No 1 647 229) following manufacturer'sinstructions for adherent cells.

[0253] As shown in FIG. 5, PDGF stimulated growth of aortic smoothmuscle cells in agreement with earlier reports (Ross et al., Philos.Trans. R. Soc. Lond. B Biol. Sci. 12: 155-169 [1990]). Pre-treatment ofcells with ErbB4-IgG immunoadhesin reduced the extent of PDGF-stimulatedproliferation of cells. Control cells treated with buffer in place ofErbB4-IgG did not show any significant effect on cell proliferation.These data indicate that at least part of the mitotic response of smoothmuscle cells is mediated by the activation of the ErbB4 receptor, andremoval of ligands which would activate the ErbB4 receptor with theErbB4 immunoadhesin reduces smooth muscle cell proliferation in responseto PDGF.

Example 3 Effect of ErbB4-IgG Immunoadhesin on Human Aortic SmoothMuscle Cell Migration

[0254] Human aortic smooth muscle cells were trypsinized and resuspendedat a concentration of 5×10⁵ cells per ml in DME containing 10% FBS.Cells were preincubated with Her4-IgG (400 nM) or buffer for 15 min. Thelower wells of ChemoTX chemotaxis chambers (Neuroprobe Inc., Cat 116-8)were filled with 300 μl of a solution of 2 U/ml human thrombin or buffer(PBS) negative control. A filter was mounted on top of the chamber andthe smooth muscle cells (buffer or ErbB4 treated) were added to the topwells in a volume of 50 μl. The plate and filter were covered with theclear plastic lid and incubated for 3 h at 37° C. in humidified air with5% CO₂. At the end of the incubation, filters were removed and the topsides were wiped with a Q-tip to remove any remaining cells. The filterswere stained with Dif-Quick staining solution and the number of cellsmigrated to the bottom of the filter were counted using an invertedphase microscope. Six wells in each group and 40 fields in each wellwere counted.

[0255] As shown in FIG. 6, thrombin acted as a chemotactic stimulus andinduced migration of aortic smooth muscle cells. ErbB4-IgG immunoadhesininhibited thrombin-stimulated cell migration. These data indicate thatat least part of thrombin's ability to stimulate smooth muscle cellmigration is mediated by the release of ligand(s) for the ErbB4receptor, and that the removal of these ligands with the ErbB4immunoadhesin reduces the chemotactic response to thrombin

Example 4 Production and Characterization of Anti-ErbB4 MonoclonalAntibodies

[0256] Generation of anti-ErbB4 Mabs

[0257] A panel of 34 murine monoclonal antibodies which specificallybind the extracellular domain of ErbB4 were produced using conventionalhybridoma technology (Table 2). Total cellular RNA was extracted fromMDA-MB-453 cells and used as a template in RT PCR to generate the humanErbB4 extracellular domain (ECD) coding sequence. Specificoligonucleotides used in the RT PCR reactions were synthesized on thebasis of the ErbB4 DNA sequence. A gDErbB4 ECD fusion protein wasconstructed by ligating the coding sequences for amino acids 1-52 ofherpes simplex virus type I glycoprotein D to the sequences encodingamino acids 26-640 of human ErbB4. The gDErbB4 ECD cDNA was insertedinto the cytomegalovirus-based expression vector pRK5. This constructwas transiently transfected into human embryonic kidney 293 cells usinga standard calcium phosphate precipitation protocol.

[0258] An affinity column was prepared by coupling the anti-gDmonoclonal 5B6 to CNBR sepharose (Pharmacia LKB Biotechnology, UppsalaSweden). Supernatant from gDErbB4 ECD transfected 293 cells wasconcentrated 20-40 fold on a ym30 membrane (Amicon, Beverly Mass.) andloaded onto the affinity resin. The column was washed with PBS and thereceptor was eluted with 100 mM acetic acid 500 mM NaCl pH 2.4. TheErbB4 ECD was buffer exchanged into PBS and concentrated. Proteinconcentration was determined by 00280.

[0259] Balb/c mice were immunized with approximately 5 g of ErbB4 ECD inRIBI MPL+TDM+CWS Emulsion (RIBI ImmunoChem Research Inc., Hamilton,Mont.) in their rear footpads on weeks 0, 1, 2 and 3. The immunized micewere tested for an antibody response by ELISA. The mice with the highesttiters were given an additional 5 g of ErbB4 ECD in RIBI during week 4.Three days later, the lymphocytes from the popliteal and inguinal nodeswere fused with mouse myeloma line X63-Ag8.653. Fused cells were platedat a density of 200,000 cells per well in 96-well tissue culture platesand hybridoma selection using HAT media supplement (Sigma, St. Louis,Mo.) began one day post fusion. Beginning on day 10, the hybridomasupernatants were screened for the presence of ErbB4 specific antibodiesusing a radioactive capture assay as described below. Stable antibodyproducing clones were obtained by limiting dilution and large quantitiesof specific Mabs were produced in ascites. The antibodies were purifiedon protein A-Sepharose columns (Fermentech, Inc., Edinburgh, Scotland)and stored sterile in PBS at 4° C.

[0260] In the radioactive capture assay, Maxisorp breakapart modules(Nunc, Roskilde, Denmark) were coated with 100 l of 2 g/ml goatanti-mouse IgG (Boehringer Mannheim) overnight at 4 C. The plates werewashed with PBS/0.5% Tween 20 (PBST), blocked with ELISA diluent(PBS/0.5% BSA/0.05% Tween 20) and incubated with monoclonal supernatantsfor 2 hr at ambient temperature. The plates were washed and incubatedfor an additional hour with 40,000 counts/well of [¹²⁵I]ErbB4 ECD. Afterwashing, the amount of ErbB4 bound to the antibodies was determined bycounting the wells on a Wallac 1277 GammaMaster (Wallac Inc,Gaithersburg, Md.).

[0261] The 34 anti-ErbB4 monoclonal antibodies produced by this method(Table 2) have a high affinity for the receptor, exhibit a diversity ofisotypes and are directed to 18 distinct epitopes on the ErbB4 ECD.Isotypes of the antibodies were determined using a Mouse MonoAb ID/SPisotyping kit from Zymed (So. San Francisco, Calif.), followingsupplier's instructions.

[0262] Testing the Specificity of Anti-ErbB4 Antibodies

[0263] The specificity of the Mabs was determined in an ELISA measuringtheir ability to bind immobilized HER2, HER3 and ErbB4 extracellulardomains (amino acids 1-645, 1-617 and 1-640 respectively). ECDs werecoated on ELISA plates at a concentration of 1 g/ml and incubated withbiotinylated anti-ErbB4 Mabs. Bound Mabs were detected usingstreptavidin peroxidase (Sigma, St. Louis, Mo.) and the substrate OPD(Sigma, St. Louis, Mo.). As can be seen in Table 2, nearly all of theantibodies produced were highly specific for ErbB4 (indicated by a ‘4’in the column labeled ‘Specificity’). Four of the antibodies showed somebinding to HER3 (indicated by a ‘3’ in the column labeled‘Specificity’).

[0264] Epitope Mapping and Characterization

[0265] The ErbB4 epitope bound by each of the monoclonal antibodies wasdetermined by competitive binding analysis (Fendly et al. CancerResearch 50:1550-1558 (1990)). The anti-ErbB4 Mabs were diluted to aconcentration of 25 g/ml in ELISA diluent and 50 l was added to an ELISAplate precoated with gDErbB4 ECD as above. The plates were incubated atroom temperature for 2 hours and washed with PBST. Dilutions ofbiotinylated anti-ErbB4 antibodies ranging from 1:1,000 to 1:10,000 wereprepared and 50 l was added to the assay plate. Following a one-hourroom temperature incubation, the plates were washed and 50 l of a 1:5000dilution of streptavidin peroxidase (Sigma) was added. The plates weredeveloped using OPD (Sigma). The anti-ErbB4 Mabs were grouped intoepitopes based on their ability to block binding of the others by 50% orgreater in comparison to an irrelevant Mab control. The relative epitopemapping identified 17 distinct epitopes, identified in Table 2 as A-Q.

[0266] The activities of nine representative antibodies wereinvestigated further. TABLE 2 Summary table of anti-ErbB4 monoclonalsMab Isotype Epitope Specificity 4-1440 IgG2b, κ B 4 4-1441 IgG1, κ J 44-1459 IgG2a, κ D 4 4-1460 IgG1, κ C 4 4-1461 IgG2a, κ E 4 4-1462 IgG1,κ C 4 4-1463 IgG2a, κ D 4 4-1464 IgG2b, κ C 4 4-1465 IgG2a, κ L 3, 44-1472 IgG2a, κ M 4 4-1473 IgG2a, κ F 4 4-1474 IgG2b, κ G 4 4-1475IgG2b, κ P 4 4-1476 IgG2a, κ K 4 4-1477 IgG2a, κ Q 4 4-1478 IgG2a, κ I 44-1479 IgG2a, κ D 4 4-1481 IgG2a, κ H 3, 4 4-1482 IgG2b, κ H 4 4-1483IgG1, κ R 3, 4 4-1484 IgG1, κ E 4 4-1485 IgG2a, κ F 4 4-1491 IgG2a, κ G4 4-1492 IgG2b, κ A 4 4-1493 IgG2B, κ A 4 4-1494 IgG2b, κ B 4 4-1495IgG2b, κ A 4 4-1496 IgG1, κ A 3, 4 4-1497 IgG1, κ N 4 4-1498 IgG2b, κ E4 4-1535 IgG2b, κ B 4 4-1536 IgG2b, κ A 4 4-1537 IgG2b, κ B 4 4-1543IgG2a, κ O 4

[0267] Determination of Binding Affinity

[0268] The relative affinities of the anti-ErbB4 Mabs were determinedaccording to the method described by Friguet et al. (J Immunol Methods.77(2):305-19 (1985)). Various concentrations of the ErbB4 ECD (1.1×10⁻⁷M to 1.08×10⁻¹⁰ M) were mixed with a constant concentration ofanti-ErbB4 Mab (2.08×10⁻¹⁰ M) and incubated overnight at 4° C. Followingincubation, the unbound Mabs were assayed by adding 100 l of thereaction mixture in duplicate to microtiter plates (Nunc) previouslycoated with gDErbB4 ECD (100 l/well at a concentration of 1 g/ml in0.05M carbonate buffer, pH 9.6 for 16 hr at 4° C.) and incubated for 1hour at room temperature. After washing with PBST, the bound Mabs weredetected by adding 100 l/well of a 1:5000 dilution of goat anti-mouseF(ab′)₂ peroxidase (Boehringer Mannheim) for one hour at roomtemperature. The plates were developed using o-phenylenediaminedihydrochloride substrate (OPD, Sigma, St. Louis, Mo.) and read on aplatereader.

[0269] The Mabs all showed high affinity binding, with Kd's ranging from0.4 to 12 nm as presented in Table 3.

[0270] Non-Reducing Immuoblot

[0271] The ability of the anti-ErbB4 Mabs to bind reduced and nonreducedErbB4 ECD was tested by immunoblot analysis. ErbB4 ECD was added totricine sample buffer, with and without BME, and applied to a 10-20%Novex tricine gel (Novex, San Diego, Calif.). The gel was run at 100Vand electroblotted for 60 min. at 0.5 amp onto a PVDF, Immobilon P,membrane (Millipore, Bedford Mass.). The membrane was washed with PBSTand blocked overnight with PBS/0.5% BSA/0.1% Tween 20, and incubatedwith 1 g/ml monoclonal antibody for 1.5 hour at ambient temperature. Themembrane was washed and incubated for an additional hour with a 1:10,000dilution of rat anti-mouse IgG peroxidase (Boehringer Mannheim). Themembrane was washed thoroughly and developed using the Amersham ECLchemiluminescence system (Amersham Life Science Inc., Arlington Heights,Ill.).

[0272] None of the Mabs were able to recognize reduced ErbB4 ECD (datanot shown), suggesting that they are directed to conformationalepitopes. Mabs identified as positive in Table 3 are those that are ableto recognize low concentrations of non-reduced ErbB4 ECD. Mabs 4-1459,4-1460, 4-1461, 4-1462, 4-1492 and 4-1497 demonstrated a high level ofimmunoreactivity and were able to bind non-reduced ErbB4 ECD at levelsdown to 0.3 ng.

[0273] Inhibition of HRG Binding

[0274] A K562 cell line that does not express any EGFR-like receptorswas used to further characterize the anti ErbB4 monoclonal antibodies. AK562 cell line transfected with ErbB4 (1E10.1H4) was produced andcultured in RPMI 1640 with 2 mM L-glutamine (GIBCO/BRL), 10% FBS(Hyclone) and 800 g/ml Geneticin, G418 (Gibco/BRL). At least 20 hr priorto assay, 1E10.1H4 was stimulated with 10 nm phorbol-12-myristate,13-acetate (PMA, Calbiochem, La Jolla Calif.). The anti-ErbB4 Mabs wereevaluated for their ability to block the binding of HRG to this cellline.

[0275] Quadruplicate samples containing 1.0×10⁵ K562 ErbB4 cellsresuspended in 200 l of RPMI 1640 with 10 mM HEPES and 0.1% BSA (bindingbuffer) were incubated with 132 pM [¹²⁵I]HRG 1₍₁₇₇₋₂₄₄₎, in the presenceof 100 nM anti-ErbB4 Mabs, overnight on ice. Following incubation, thecells were collected using a Multiscreen filtration device (Millipore),and washed twice with 200 l ice cold binding buffer. Cell associatedcounts were measured on a gamma counter. The percent binding wascalculated against a control sample containing no Mab. The nonspecificbinding was determined by incubation of a sample in the presence of 500nM cold HRG 1₍₁₇₇₋₂₄₄₎. Mabs were considered positive for HRG blockingif they blocked 90% or greater binding. As can be seen in Table 3, sixof the nine anti-ErbB4 antibodies tested were able to inhibit ¹²⁵I-HRGbinding at this level. Mab 4-1461 inhibited binding by 7% and 1459exhibited no HRG blocking. The anti-ErbB4 Mab 4-1497 did not inhibitbinding but rather appeared to enhance HRG binding by 26%.

[0276] Inhibition of HRG Binding in Human Breast Cancer Cell Lines

[0277] Since a number of the anti-ErbB4 Mabs were able to block bindingof HRG to transfected K562 cells, their ability to block HRG binding toseveral human mammary carcinoma cell lines was tested. The cell linesMDA-MB-453, T470 and BT474 (ATCC, Rockville, Md.) were plated into 24well tissue culture plates at a density of 1×10⁵ cells per well andallowed to adhere overnight. The anti-ErbB4 Mabs or anti-HER-2 controlMabs 2C4 and 4D5 were diluted to a concentration of 100 nM in Ham's F-12plus Dulbecco's modified Eagle medium (1:1, v/v) with 10 mM HEPES and0.1% BSA (binding buffer) and added in triplicate to the plates.Following a 30 minute incubation on ice, 1.5×10⁵ counts of [¹²⁵I] HRG1₍₁₄₄₋₂₇₇₎ was added. The plates were incubated on ice for four hoursand washed twice with ice cold binding buffer. The cells weresolubilized with 8 M urea/3 M acetic acid and cell associated countswere measured on a Wallac 1277 GammaMaster. The percent binding wascalculated as above. The nonspecific binding was determined byincubation of a sample in the presence of 100 nM cold HRG 1₍₁₄₄₋₂₇₇₎.

[0278] None of the anti-ErbB4 Mabs caused significant inhibition of¹²⁵IHRG binding to the carcinoma lines tested. In contrast, theanti-HER-2 control Mabs 2C4 and 4D5 blocked binding by 84% and 29%respectively in MDA-MB-453 cells, 70% and 48% in T470 cells and 57% and12% in BT474 cells. The unlabeled HRG control blocked 99% binding inMDA-MB 453 cells, 98% binding in T47D cells and 96% binding in BT474cells at a concentration of 100 nM. This data suggests that in thesecell lines the ErbB4 receptor may play a minor role in mediating the HRGresponses.

[0279] Inhibition of Tyrosine Phosphorylation

[0280] Heregulins have been shown to induce the tyrosine phosphorylationof ErbB4. Therefore it was of interest to determine if the anti-ErbB4Mabs were able to affect HRGβ1_((177,244)) stimulated phosphorylation ofthe receptor in the K562 ErbB4 cell line.

[0281] The ErbB4 transfected K562 cell line (1E10.1H4) was grown in RPMI1640 culture media to a density of 1×10⁶ cells/ml. The cells were thenchanged to serum-free media without PMA (assay buffer) and incubated at37° C. for 2-6 hours. The cells were washed with assay buffer andduplicate samples containing 2.5×10⁵ cells in assay buffer with 0.1%BSA, were incubated with 25 ug of anti-ErbB4 Mabs or a control Mab for30 min. at room temperature. Following incubation, one set of thesamples was stimulated with 15 mM HRG 1₍₁₇₇₋₂₄₄₎ for 8 minutes at roomtemperature. The supernatants were removed and the cells lysed for 5minutes at 100° C. in 100 l of SDS sample buffer containing 50l/ml-mercaptoethanol. A 30 l aliquot of each sample was electrophoresedin a 4-12% polyacrylamide gel (Novex) and electroblotted onto a PVDFmembrane (Millipore). The membranes were blocked with 2% BSA intris-buffered saline containing 0.05% Tween-20 overnight at 4° C. andincubated with a 1:1000 dilution of recombinant antiphosphotyrosineperoxidase monoclonal RC20H (Transduction Laboratories, Lexington Ky.)for 4 hours at room temperature. Bound anti-phosphotyrosine Ab wasvisualized using the Amersham ECL system (Amersham Life Science Inc.)and quantified by densitometry.

[0282] Six of nine monoclonal antibodies tested inhibited the generationof an HRG-induced tyrosine phosphorylation signal (Table 31. Theremaining three were not inhibitory and none of the anti-ErbB4 Mabs wasable to stimulate phosphorylation of the ErbB4 receptor.

[0283] Immunohistochemistry

[0284] Since anti-ErbB4 Mabs may be useful as diagnostic reagents, theirability to stain frozen cell pellets using standard immunocytochemicaltechniques was investigated. ErbB4 transfected K562 cells (1E10.1H4) andthe human breast carcinoma lines MDA-MB-453, T470, and BT474 (ATCC,Rockville, Md.) were pelleted and frozen in OCT compound (Miles Inc.,Elkhart, Ind.). The frozen pellets were sectioned on a cryostat to athickness of 5 microns, mounted on slides, fixed in cold acetone (4 C)for 3-5 min. and air-dried. Endogenous peroxidase activity was quenchedusing a modification of the glucose oxidase method. The slides wererinsed with PBS and the cells were blocked for endogenous biotinactivity using a Vector Biotin blocking kit (Vector, Burlingame,Calif.). Endogenous immunoglobulin binding sites were blocked with 10%normal horse serum (Vector). The cells were then incubated with 10 g/mlanti-ErbB4 Mabs for one hour at RT, followed by a 30 minute incubationwith a 1:200 dilution of biotinylated horse anti-mouse IgG (Vector). Theslides were incubated with ABC Elite Reagent (Vector) for 30 min. andthe ErbB4 receptors visualized using DAB (Pierce, Rockford, Ill.).Mayer's hematoxylin (Rowley Biomedical Institute, Rowley, Mass.) wasused to counterstain the cells.

[0285] Many of the anti-ErbB4 Mabs were able to stain the ErbB4transfected K562 cells with varying intensity and little or nobackground staining (Table 3). Numbers represent the intensity ofstaining compared to an irrelevant control. None of the Mabs was able tostain the frozen human mammary carcinoma cells that were tested (datanot shown). TABLE 3 Summary table of monoclonal antibody activityNon-Reducing HRG P-Tyr Histo- Mab Isotype Epitope Kd(nM) ImmunoblotBlocking Blocking chemistry 4-1440 IgG2b, κ B 1.9 − + + 3+ 4-1459 IgG2a,κ D 0.7 + − − 4+ 4-1460 IgG1, κ C 1.2 + + + 3+ 4-1461 IgG2a, κ B 2.3 + −− 4+ 4-1462 IgG1, κ C 0.4 + + + 2+ 4-1464 IgG2b, κ C 1.0 − + + 2+ 4-1473IgG2a, κ F 6.0 − + + 2-3+ 4-1492 IgG2b, κ A 2.1 + + + − 4-1497 IgG1, κ N12.0 + − − −

[0286] FACS Analysis

[0287] To determine whether the anti-ErbB4 Mabs could bind to ErbB4 onthe surface of viable cells, FACS analysis was done using the ErbB4transfected K562 cell line and the mammary carcinoma lines MDA-MB-453,T47D and BT-474. Adherent cells were detached from tissue culture flasksusing 10 mM EDTA in PBS, centrifuged at 1400 rpm for 5 min. andresuspended in PBS with 1% fetal bovine serum (FACS diluent). The cellswere counted, adjusted to 10⁷ cells/ml and 0.1 ml of cells was incubatedwith 10 g/ml of each Mab in 100 l FACS diluent for 30 min. at 4° C. Thesamples were washed, resuspended in 0.1 ml diluent and incubated with 1g of FITC conjugated F(ab′)₂ fragment of goat anti-mouse IgG (BoehringerMannheim) for 30 min at 4° C. The cells were washed, resuspended in 0.5ml FACS diluent and analyzed using a FACScan cell sorter (BectonDickinson, Mt. View, Calif.). Data was gated by forward and side scatterand propidium iodide fluorescence to exclude debris, doublets and deadcells.

[0288] All of the Mabs bound to the ErbB4 receptor on the ErbB4transfected K562 cell line, which is expressed at approximately 2×10⁵receptors/cell. An increase in observed cellular fluorescence of theErbB4 transfected K562 cells from 2 to 50 fold was observed whencompared to the isotype controls. Some of the weaker binding may reflecta ErbB4 ECD epitope that is sequestered on the intact cells. Incontrast, the anti-ErbB4 antibodies 4-1440, 4-1464 and 4-1492, whichgive the highest fluorescence intensity on the transfected cell line,showed minimal binding to the breast carcinoma lines MDA-MB-453, T47Dand BT-474. The positive control anti-HER2 Mab 2-2C4 showed binding tothe tumor lines in proportion to the level of HER-2 expression. Theseresults indicate a level of ErbB4 expression on the MDA-MB-453, T470 andBT-474 cells which is below the detection limit of this assay.

[0289] Inhibition of Heregulin Binding to ErbB4 Immunoadhesin

[0290]FIG. 7 shows a displacement curve of ¹²⁵IHRG binding to a ErbB4immunoadhesin captured on breakapart modules using the indicatedconcentrations of the anti-ErbB4 Mabs 4-1440, 4-1460, and 4-1464.Maxisorp breakapart modules (Nunc) were coated with 100 l of a 1:200dilution of goat anti-human Ig (Boehringer Mannheim) in 50 mM carbonatebuffer pH 9.6 overnight at 4° C. The plates were washed with PBST,blocked with ELISA diluent and incubated with 100 l of 200 ng/ml ErbB4immunoadhesin for 2 hr at ambient temperature. The plates were washedand 50 l of diluted Mabs (0.1 to 100 nM final) and 50 l of ¹²⁵I-HRG1₍₁₇₇₋₂₄₄₎ diluted to give a final concentration of 132 pM were added tothe plate. Following a 1.5 hr incubation at ambient temperature, theplates were washed and the amount of ¹²⁵IHRG bound to the receptor wasdetermined by counting the wells on a Wallac 1277 GammaMaster.

[0291]FIG. 7 demonstrates that the Mabs inhibited heregulin binding tothe immunoadhesin in a dose dependent manner with ED₅₀ values rangingfrom 0.7 to 1.1 nM. This indicates that the Mabs posses a high degree ofblocking ability.

[0292] Deposit of Material

[0293] The following hybridomas have been deposited with the AmericanType Culture Collection, 10801 University Blvd., Manassas, Va.20110-2209, USA (ATCC): Hybridoma ATCC Dep. No. Deposit DateHER4.10H1.1A1 PTA-2828 Dec. 19, 2000 HER4.1C6.A11 PTA-2829 Dec. 19, 2000HER4.3B9.2C9 PTA-2826 Dec. 19, 2000 HER4.1A6.5B3 PTA-2827 Dec. 19, 2000HER4.8B1.2H2 PTA-2825 Dec. 19, 2000

[0294] Each of the deposited hybridomas produces one of the anti-ErbB4monoclonal antibodies identified in Table 2. HER4.10H1.1A1 produces mAb4-1464, HER4.1C6.A11 produces mAb 4-1440, HER4.3B9.2C9 produces mAb4-1460, HER4.1A6.5B3 produces mAb 4.1492 and HER4.8B1.2H2 produces mAb4-1473

[0295] The deposit of the hybridomas with the ATCC was made under theprovisions of the Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purpose of Patent Procedure andthe Regulations thereunder (Budapest Treaty). This assures maintenanceof a viable culture of the deposit for 30 years from the date ofdeposit. The deposit will be made available by ATCC under the terms ofthe Budapest Treaty, and subject to an agreement between Genentech, Inc.and ATCC, which assures permanent and unrestricted availability of theprogeny of the culture of the deposit to the public upon issuance of thepertinent U.S. patent or upon laying open to the public of any U.S. orforeign patent application, whichever comes first, and assuresavailability of the progeny to one determined by the U.S. Commissionerof Patents and Trademarks to be entitled thereto according to 35 U.S.C.§ 122 and the Commissioner's rules pursuant thereto (including 37 C.F.R.§ 1.14 with particular reference to 886 OG 638).

[0296] The assignee of the present application has agreed that if aculture of the materials on deposit should die or be lost or destroyedwhen cultivated under suitable conditions, the materials will bepromptly replaced on notification with another of the same. Availabilityof the deposited material is not to be construed as a license topractice the invention in contravention of the rights granted under theauthority of any government in accordance with its patent laws.

[0297] The foregoing written specification is considered sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

What is claimed is:
 1. A method for controlling excessive proliferation or migration of smooth muscle cells comprising treating said smooth muscle cells with an effective amount of an antagonist of a native ErbB4 receptor.
 2. The method of claim 1 wherein the control is prevention of excessive proliferation or migration of smooth muscle cells.
 3. The method of claim 1 wherein the control is inhibition of excessive proliferation or migration of smooth muscle cells.
 4. The method of claim 3 wherein said inhibition is total inhibition.
 5. The method of claim 1 wherein said smooth muscle cells are pyloric smooth muscle cells.
 6. The method of claim 1 wherein said smooth muscle cells are urinary bladder smooth muscle cells.
 7. The method of claim 1 wherein said smooth muscle cells are those of an airway passage.
 8. The method of claim 1 wherein said excessive proliferation or migration of smooth muscle cells results in stenosis.
 9. The method of claim 1 wherein said smooth muscle cells are vascular smooth muscle cells.
 10. The method of claim 9 wherein said vascular smooth muscle cells are human.
 11. The method of claim 9 wherein said vascular smooth muscle cells are human aortic smooth muscle cells.
 12. The method of claim 9 wherein said excessive proliferation or migration of smooth muscle cells results in vascular stenosis.
 13. The method of claim 12 wherein said vascular stenosis is further characterized by excessive proliferation or migration of endothelial cells.
 14. The method of claim 13 wherein said stenosis is restenosis.
 15. The method of claim 1 wherein the ErbB4 receptor antagonist is an immunoadhesin.
 16. The method of claim 15 wherein said immunoadhesin comprises an extracellular domain sequence of a native ErbB4 receptor.
 17. The method of claim 16 wherein said native ErbB4 receptor is human.
 18. The method of claim 17 wherein the native human ErbB4 receptor extracellular domain sequence is fused to an immunoglobulin heavy chain constant region sequence.
 19. The method of claim 18 wherein said immunoglobulin is of IgG isotype.
 20. The method of claim 19 wherein said immunoglobulin is of IgG1, IgG2 or IgG3 isotype.
 21. The method of claim 19 wherein said immunoadhesin comprises at least one IgG immunoglobulin light chain.
 22. The method of claim 1 wherein said antagonist is an antibody.
 23. The method of claim 22 wherein said antibody is a neutralizing antibody against a native ErbB4 receptor.
 24. The method of claim 23 wherein said antibody is a chimeric, humanized or human antibody.
 25. The method of claim 23 wherein said antibody is glycosylated.
 26. The method of claim 23 wherein said antibody binds essentially the same epitope as an antibody produced by a hybridoma selected from the group consisting of HER4.10H1.1A1 (ATCC Accession Number PTA-2828), HER4.1C6.A11 (ATCC Accession Number PTA-2829), HER4.3B9.2C9 (ATCC Accession Number PTA-2826), HER4.1A6.5B3 (ATCC Accession Number PTA-2827) and HER4.8B1.2H2 (ATCC Accession Number PTA-2825).
 27. The method of claim 23 wherein said antibody has complementarity determining region (CDR) residues from an antibody produced by a hybridoma selected from the group consisting of HER4.10H1.1A1 (ATCC Accession Number PTA-2828), HER4.1C6.A11 (ATCC Accession Number PTA-2829), HER4.3B9.2C9 (ATCC Accession Number PTA-2826), HER4.1A6.563 (ATCC Accession Number PTA-2827) and HER4.8B1.2H2 (ATCC Accession Number PTA-2825).
 28. A method for treating stenosis in a mammalian patient comprising administering to said patient an effective amount of an antagonist of a native mammalian ErbB4 receptor.
 29. The method of claim 28 wherein said patient is human.
 30. The method of claim 29 wherein said stenosis is vascular stenosis.
 31. The method of claim 30 wherein said vascular stenosis is restenosis.
 32. The method of claim 28 wherein said antagonist is an immunoadhesin.
 33. The method of claim 32 wherein said immunoadhesin comprises an extracellular domain sequence of a native human ErbB4 receptor.
 34. The method of claim 33 wherein said extracellular domain sequence is fused to an immunoglobulin heavy chain constant region sequence.
 35. The method of claim 34 wherein said immunoglobulin is of IgG isotype.
 36. The method of claim 28 wherein said antagonist is an antibody.
 37. The method of claim 36 wherein said antibody is a neutralizing antibody against a native human ErbB4 receptor.
 38. The method of claim 36 wherein said antibody binds essentially the same epitope as an antibody produced by a hybridoma selected from the group consisting of HER4.10H1.1A1 (ATCC Accession Number PTA-2828), HER4.1C6.A11 (ATCC Accession Number PTA-2829), HER4.3B9.2C9 (ATCC Accession Number PTA-2826), HER4.1A6.5B3 (ATCC Accession Number PTA-2827) and HER4.8B1.2H2 (ATCC Accession Number PTA-2825).
 39. The method of claim 36 wherein said antibody has complementarity determining region (CDR) residues from an antibody produced by a hybridoma selected from the group consisting of HER4.10H1.1A1 (ATCC Accession Number PTA-2828), HER4.1C6.A11 (ATCC Accession Number PTA-2829), HER4.3B9.2C9 (ATCC Accession Number PTA-2826), HER4.1A6.5B3 (ATCC Accession Number PTA-2827) and HER4.8B1.2H2 (ATCC Accession Number PTA-2825).
 40. The method of claim 28 wherein said antagonist is administered as an injection or infusion.
 41. The method of claim 28 wherein said treatment additionally reduces hypertension associated with said stenosis.
 42. The method of claim 28 wherein said treatment is prevention.
 43. The method of claim 28 wherein said stenosis is pyloric stenosis.
 44. The method of claim 28 wherein said stenosis is thickening of the urinary bladder wall.
 45. The method of claim 28 wherein said stenosis is part of an obstructive airway disease.
 46. A method for treating stenosis in a mammalian patient comprising introducing into a cell of said patient a nucleic acid encoding an antagonist of an ErbB4 receptor.
 47. The method of claim 46 wherein said patient is human.
 48. The method of claim 47 wherein said antagonist is an immunoadhesin.
 49. The method of claim 48 wherein said immunoadhesin comprises an extracellular domain sequence of a native human ErbB4 receptor fused to an immunoglobulin heavy chain constant region sequence.
 50. The method of claim 47 wherein said antagonist is an antibody.
 51. The method of claim 50 wherein said antibody is a neutralizing antibody against a native ErbB4 receptor.
 52. The method of claim 51 wherein said antibody is a chimeric, humanized or human antibody.
 53. The method of claim 51 wherein said antibody binds essentially the same epitope as an antibody produced by a hybridoma selected from the group consisting of HER4.10H1.1A1 (ATCC Accession Number PTA-2828), HER4.1C6.A11 (ATCC Accession Number PTA-2829), HER4.3B9.2C9 (ATCC Accession Number PTA-2826), HER4.1A6.5B3 (ATCC Accession Number PTA-2827) and HER4.8B1.2H2 (ATCC Accession Number PTA-2825).
 54. The method of claim 51 wherein said antibody has complementarity determining region (CDR) residues from an antibody produced by a hybridoma selected from the group consisting of HER4.10H1.1A1 (ATCC Accession Number PTA-2828), HER4.1C6.A11 (ATCC Accession Number PTA-2829), HER4.3B9.2C9 (ATCC Accession Number PTA-2826), HER4.1A6.5B3 (ATCC Accession Number PTA-2827) and HER4.8B1.2H2 (ATCC Accession Number PTA-2825).
 55. The method of claim 46 wherein said nucleic acid is introduced in vivo.
 56. The method of claim 46 wherein said nucleic acid is introduced ex vivo.
 57. A method for treating hypertension associated with vascular stenosis in a mammalian patient, comprising administering to said patient an effective amount of an antagonist of a native mammalian ErbB4 receptor.
 58. The method of claim 57 wherein said antagonist is a small molecule.
 59. A pharmaceutical composition for the treatment of stenosis in a mammalian patient comprising an effective amount of an antagonist of a native mammalian ErbB4 receptor, in admixture with a pharmaceutically acceptable carrier.
 60. A method for identifying a molecule that inhibits or enhances the proliferation or migration of smooth muscle cells, comprising the steps of: (a) contacting a polypeptide comprising an amino acid sequence having at least 85% sequence identity with the amino acid sequence of the extracellular domain of a native ErbB4 receptor and retaining the ability to control excessive proliferation or migration of smooth muscle cells, with a candidate molecule; and (b) determining whether the candidate molecule inhibits or enhances the ability of said polypeptide to control excessive proliferation or migration of smooth muscle cells.
 61. The method of claim 60 wherein said polypeptide comprises the extracellular domain of a native ErbB4 receptor.
 62. The method of claim 61 wherein said receptor is human.
 63. The method of claim 61 wherein said polypeptide is an immunoadhesin.
 64. The method of claim 60 wherein said molecule enhances the ability of said polypeptide to control excessive proliferation or migration of smooth muscle cells.
 65. The method of claim 64 wherein said molecule is selected from the group consisting of antibodies and small molecules.
 66. An antibody that binds essentially the same epitope of ErbB4 as an antibody produced by a hybridoma selected from the group consisting of HER4.10H1.1A1 (ATCC Accession Number PTA-2828), HER4.1C6.A11 (ATCC Accession Number PTA-2829), HER4.3B9.2C9 (ATCC Accession Number PTA-2826), HER4.1A6.5B3 (ATCC Accession Number PTA-2827) and HER4.8B1.2H2 (ATCC Accession Number PTA-2825).
 67. An antibody that has complementarity determining region (CDR) residues from an antibody produced by a hybridoma selected from the group consisting of HER4.10H1.1 A1 (ATCC Accession Number PTA-2828), HER4.1C6.A11 (ATCC Accession Number PTA-2829), HER4.3B9.2C9 (ATCC Accession Number PTA-2826), HER4.1A6.5B3 (ATCC Accession Number PTA-2827) and HER4.8B1.2H2 (ATCC Accession Number PTA-2825).
 68. An antibody selected from the group consisting of an antibody produced by a hybridoma selected from the group consisting of HER4.10H1.1A1 (ATCC Accession Number PTA-2828), HER4.1C6.A11 (ATCC Accession Number PTA-2829), HER4.3B9.2C9 (ATCC Accession Number PTA-2826), HER4.1A6.5B3 (ATCC Accession Number PTA-2827) and HER4.861.2H2 (ATCC Accession Number PTA-2825).
 69. An antibody that binds essentially the same epitope of ErbB4 bound by an antibody selected from the group consisting of anti-ErbB4 monoclonal antibodies 4-1440, 4-1460, 4-1473, 4-1492 and 4-1464.
 70. An antibody that has complementarity determining region (CDR) residues from an antibody selected from the group consisting of anti-ErbB4 monoclonal antibodies 4-1440, 4-1460, 4-1473, 4-1492 and 4-1464.
 71. An antibody which binds to ErbB4 with high affinity.
 72. The antibody of claim 71 which binds to ErbB4 with a Kd of less than 100 nM.
 73. The antibody of claim 71 which binds to ErbB4 with a Kd of less than 50 nM.
 74. The antibody of claim 71 which binds to ErbB4 with a Kd of less than 10 nM.
 75. The antibody of claim 71 which is a humanized antibody.
 76. The antibody of claim 71 which is a human antibody.
 77. The antibody of claim 71 which is an antibody fragment.
 78. An antibody which is capable of binding to both ErbB4 and ErbB3.
 79. The antibody of claim 78 which binds ErbB4 with high affinity.
 80. The antibody of claim 78 which binds both ErbB4 and ErbB3 with high affinity.
 81. An antibody which binds to ErbB4 and reduces heregulin binding thereto.
 82. The antibody of claim 81 which binds ErbB4 with high affinity.
 83. An antibody which binds to ErbB4 and reduces heregulin-induced tyrosine phosphorylation thereof.
 84. The antibody of claim 83 which binds ErbB4 with high affinity. 